title: (T33_MIL_5733) Group 2, TFH cells ICOS-L blockade, Exp. 2

status: Public on Jan 19 2015

description: Gene expression profiles of antigen-specific TFH cells prepared from the draining lymph nodes of C57BL/6 mice were compared using Affymetrix MG 430 2.0 GeneChip arrays. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized in duplicates for each of the two groups to the GeneChip arrays. Two chips in two groups were hybridized and analyzed: Group 2 ICOS L blockade group.

hyb_protocol: The MG430Av2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

data_processing: Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Grün) with the BioRetis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 2 + 2 chips were compared to each other (control group vs. blockade group, 4 comparisons, and all chips within the groups, twice 2 comparisons), and the following parameters of absolute and comparative analysis were included in the BioRetis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t tests between 4 SLR values of control vs. blockade and 4 SLR values within both groups (the latter 4 giving a mean SLR value of zero; p-value had to be <= 1.386 E-07) or more than 50% of non parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 3 or more of all 4 Change calls have to be in the same direction. Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (one direction).

source_name: OT-II TFH cells from draining lymph node, day 6 after immunization, 6 h after mAb treatment. Group 2: mAb MIL-5733 (anti-ICOS L).

label_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays.

growth_protocol: 2.5 E05 ovalbumin- (OVA-) specific OT-II T cells (Thy-1.1pos) were co-transferred together with 1 E06 nitrophenol- (NP-) specific B1-8i B cells into C57BL/6 recipient mice immunized s.c. with 50 µg NP-OVA in complete Freund´s adjuvant. On day 6 after immunization, recipients were treated i.p. with 150 µg mAb MIL-5733 (anti-ICOS-L) or a rat IgG2a isotype control (clone 1D10). 6 hours later, total cells from inguinal lymph nodes (pool of 20 animals) were isolated and enriched for transgenic T cells by magnetic sorting using Thy-1.1 microbeads. TFH cells were isolated on a FACS ARIA II by sorting for DAPIneg B220neg CD4pos Thy 1.1pos CXCR5pos PD-1pos cells, resulting in a purity of > 99% Thy-1.1pos transgenic T cells and > 95% CXCR5pos PD-1pos TFH cells.

extract_protocol: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction, including the recommended DNase digest. The amount, purity, and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).

tag: recipient strain

value: draining lymph node

value: OT-II T follicular helper cells (CXCR5pos PD-1pos)

tag: treatment

value: 6 h in vivo with mAb MIL-5733


title: (T32_MIL_5733) Group 2, TFH cells ICOS-L blockade, Exp. 1

type: RNA

city: Berlin

first: Pawel

last: Durek

address: Charitéplatz 1

country: Germany

institute: Deutsches Rheuma-Forschungszentrum

scan_protocol: Arrays were scanned with an Affymetrix GeneChip Scanner 3000, using the GCOS software version 1.4 from Affymetrix.

label: Biotin

molecule: total RNA

organism: Mus musculus

tag: gender

value: female

tag: tissue

tag: cell type


title: Grp3_Chip1_CD44negCD127pos_spl_exvivo

status: Public on Feb 05 2015

description: Naive (CD44-CD127+) CD8+CD3+ T cells from murine spleen, ex vivo isolated; group 3, chip 1

hyb_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG_U430_2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

data_processing:
Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Grün) with the BioRetis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMed ID: 19265543). Chip data included in the BioRetis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All five groups were compared to every other group to obtain 10 lists of significant differentially regulated genes. All 3 chips of every group (Experiment group) were compared to the 3 chips of one of the other groups (Baseline group) and chips within one group were compared to each other in both directions. The following parameters of absolute and comparative analysis were included in the BioRetis database for all 10 comparisons: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between 9 SLR values of Experiment group vs. Baseline group and 2 times 6 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls of Experiment vs. Baseline group have to be in the same direction. Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria. These are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets, respectively and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (in one direction only; 65 true positive IDs in average).
Data tables contain the following: Probe Set ID; VALUE: GCOS Signal, i.e. gene expression height; Detection call: A: gene is detected as Absent, M: as Marginal present, or P: as Present; P-value: Non-parametrical p-value for generation of detection call

source_name: Group 3: spleen naive CD8 T cells

growth_protocol: Ex-breeder female mice were purchased from Charles-River laboratories, Sulzfeld,Germany. Single cell suspension from the spleen and bones (tibiae, femur, hips and sternum) of aged C57BL/6 mice were prepared. CD8+ T cells were first enriched with α-CD8 α microbeads (Miltenyi). Naive (CD44-CD127+) and memory (CD44+CD127+) CD8+CD3+ T cells were then cytometrically sorted with FACS Aria or Influx (BD Biosciences). The cells of 3-12 mice were pooled. Sorted cells were then either immediately lysed with RA-1 buffer (Macherey-Nagel) for RNA preparation or were activated with α-CD3 and α-CD28. For this, wells of a 96-well plate were coated with α-CD3 (2.5 µg/ml) and α-CD28 (2.5 µg/ml) at 37 °C for 2-3 hours. After the excess antibody was washed away, approximately 2.0E05 cells were plated per well. Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 1% Streptomycin, 1% Penicillin and 0.1% β-mercaptoethanol. 42-44 hours later, activated cells were lysed with RA-1 buffer.

extract_protocol: Total RNA was extracted using the NucleoSpin RNA (Macherey-Nagel) according to the manufacturer’s instruction, including the recommended DNase digestion. Contaminating genomic DNA was removed by an on-column DNA digestion step (Macherey-Nagel). The amount of RNA was checked with a NanoDrop ND-1000 spectrophotometer.

tag: strain

value: Spleen

tag: cells

value: CD8+CD3+ T cells; Naive (CD44-CD127+); 3-12 mice were pooled

tag: magnetic cell sorting

tag: flow cytometric cell sorting

value: Naive (CD44-CD127+) CD8+CD3+ T cells were cytometrically sorted with FACS Aria or Influx (BD Biosciences)

tag: activation

value: not activated; ex vivo isolated

tag: cell cultivation

value: No cultivation, immediately lysed with RA-1 buffer (Macherey-Nagel) for RNA preparation


title: Grp3_Chip3_CD44negCD127pos_spl_exvivo

description: Naive (CD44-CD127+) CD8+CD3+ T cells from murine spleen, ex vivo isolated; group 3, chip 3

label_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG_U430_2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

value: CD8+ T cells were first enriched with α-CD8 α microbeads (Miltenyi)


title: Grp1_Chip2_CD44posCD127pos_spl_exvivo

description: Memory (CD44+CD127+) CD8+CD3+ T cells from murine spleen, ex vivo isolated; group 1, chip 2

source_name: Group 1: resting spleen memory CD8 T cells

value: CD8+CD3+ T cells; Memory (CD44+CD127+); 3-12 mice were pooled

value: Memory (CD44+CD127+) CD8+CD3+ T cells were cytometrically sorted with FACS Aria or Influx (BD Biosciences)


title: IL-15 9

status: Public on Apr 07 2015

type: genomic

city: Vancouver

first: Wendy

last: Robinson

state: BC

address: 950 W 28th Ave

department: Medical Genetics

country: Canada

institute: University of British Columbia

hyb_protocol: Illumina HumanMethylation450 BeadChip Protocol

scan_protocol: Arrays were imaged using Illumina iScan on a two-color channel

data_processing: GenomeStudio 2011.1 software and R version 2.14.0; Normalization using SWAN; The file Unmethylated_and_methylated_signal.txt is available on the series record and contains the raw data.

label: Cy5 and Cy3

molecule: genomic DNA

organism: Homo sapiens

source_name: Cultured Placenta Villous explant exposed to media supplemented with IL-15

label_protocol: Cy5 and Cy3

growth_protocol: Placental tissues were collected from consenting healthy women undergoing elective pregnancy termination at gestational ages between 7-9 weeks.  dNK were isolated from decidua after collagenase and hyaluronidase digestion, depletion of attached cells, and were then enriched by human NK cell enrichment cocktail following the manufacturer’s instructions. The percentage of CD56+/CD16- dNK reached ~87%, CD16+ cells were < 2.1% (Hu et al., 2006). The dNK were trapped inside of hollow fiber (Spectrum Laboratories, Inc., Rancho Dominquez, CA, USA) (HF-dNK) and used for co-culturing with explants in order to eliminate trophoblast cell contamination from dNK when performing EVT DNA extraction. HF permits soluble factors with molecular weight below 500 kDa to be exchanged freely. For each placental specimen, villous explant cultures were set up in 12 well plates on collagen gel. After overnight incubation, explant cultures with EVT outgrowth on villous tips were identified and exposed to medium control (DMEM/F12 supplemented with 10% FBS), or IL-15 alone, or concordant HF-dNK plus IL-15 for a total of 96 hours. 6-8 explants were used for each treatment group in each placental specimen.

extract_protocol: For each of the explant cultures, EVT were harvested from collagen gel after dissecting villi tissue and pooled from each treatment group. Previous study has shown that the EVT forming the cell column and migrating on collagen were cytokeratin positive and vimentin negative cells and purity was high (Hu et al., 2006; Hu et al., 2008). Five placental specimens in total were used for global DNA methylation array and Bisulfite pyrosequencing validation. Genomic DNA from EVT was purified using QIAamp DNA mini kit  according to the manufacturer’s instructions (Qiagen Inc. Mississauga, ON, Canada).

value: Placenta villous explant

tag: trimester

value: First Trimester


title: Control 9

source_name: Cultured Placenta Villous explant


title: (T33_1D10) Group 1, TFH cells control, Exp. 2

description: Gene expression profiles of antigen-specific TFH cells prepared from the draining lymph nodes of C57BL/6 mice were compared using Affymetrix MG 430 2.0 GeneChip arrays. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized in duplicates for each of the two groups to the GeneChip arrays. Two chips in two groups were hybridized and analyzed: Group 1 control group.

source_name: OT-II TFH cells from draining lymph node, day 6 after immunization, 6 h after mAb treatment. Group 1: mAb 1D10 (isotype control)

value: 6 h in vivo with mAb 1D10


title: IL-15 5


title: Hollow Fibre 4

source_name: Cultured Placenta Villous explant exposed to decidua natural killer cells trapped in a hollow fiber


title: Control 4


title: Hollow Fibre 9


title: IL-15 1


title: Control 7


title: Control 8


title: (T32_1D10) Group 1, TFH cells control, Exp. 1


title: Control 5


title: Hollow Fibre 8


title: Control 1


title: Group2 Chip1: ND_1_BMPC_CD19P CD19 positive

status: Public on Dec 31 2014

description: Group2 Chip1 ND1: Gene-expression profiling of primary human bone marrow plasma cells expressing CD19.

hyb_protocol: The HG-U133 Plus 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

data_processing: Data were analyzed according to High Performance Chip Data Analysis (HPCDA) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 4 chips of group 1 were compared to the 4 chips of group 2 and chips within one group were compared to each other in both directions. The following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between 16 SLR values of Experiment group vs. Baseline group and 24 SLR values within both groups in both directions (these 24 giving a mean SLR value of zero; p-value had to be <= 2.286E-08) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 9 or more of all 16 Change calls have to be in the same direction. Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (one direction).

source_name: CD19-positive bone marrow plasma cells

label_protocol: Labeling of total RNA was performed using the two cycle GeneChip 3' IVT Expression Kit from Affymetrix according to the manufacturer's instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, two times amplified, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays.

extract_protocol: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instruction, including the recommended DNase digest. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).

treatment_protocol: Plasma cells were enriched magnetically from bone marrow single cell suspensions of healthy donors after lysis of erythrocytes using Qiagen EL buffer, using anti-CD138-coupled magnetic beads. Next, the enriched plasma cells were stained with fluorescently labeled monoclonal antibodies detecting CD138, CD38, CD19, CD20, CD3, and CD14 and were sorted by Aria or DIVA FACSorter machine according to expression of CD19.

value: primary bone marrow cells from healthy donors

tag: separation

value: erythrocyte lysis (Qiagen EL)

value: positive enrichment of CD138+ cells


title: Group2 Chip3: ND_3_BMPC_CD19P CD19 positive

description: Group2 Chip3 ND3: Gene-expression profiling of primary human bone marrow plasma cells expressing CD19.


title: Group2 Chip2: ND_2_BMPC_CD19P CD19 positive

description: Group2 Chip2 ND2: Gene-expression profiling of primary human bone marrow plasma cells expressing CD19.


title: Grp2_Chip1_CD44posCD127pos_bm_exvivo

description: Memory (CD44+CD127+) CD8+CD3+ T cells from murine bone marrow, ex vivo isolated; group 2, chip 1

source_name: Group 2: resting bone marrow memory CD8 T cells

value: Bone marrow


title: Group1 Chip4: ND_4_BMPC_CD19M CD19 negative

description: Group1 Chip4 ND4: Gene-expression profiling of primary human bone marrow plasma cells not expressing CD19.

source_name: CD19-negative bone marrow plasma cells


title: Group1 Chip1: ND_1_BMPC_CD19M CD19 negative

description: Group1 Chip1 ND1: Gene-expression profiling of primary human bone marrow plasma cells not expressing CD19.


title: Group1 Chip3: ND_3_BMPC_CD19M CD19 negative

description: Group1 Chip3 ND3: Gene-expression profiling of primary human bone marrow plasma cells not expressing CD19.


title: Group2 Chip4: ND_4_BMPC_CD19P CD19 positive

description: Group2 Chip4 ND4: Gene-expression profiling of primary human bone marrow plasma cells expressing CD19.


title: Group1 Chip2: ND_2_BMPC_CD19M CD19 negative

description: Group1 Chip2 ND2: Gene-expression profiling of primary human bone marrow plasma cells not expressing CD19.


title: Gene expression of murine fibroblast-like synoviocytes Grp1_C57BL6_3_FLS_TNFtg_plus_RSK2-KO

status: Public on Dec 04 2014

description: Gene-expression profiling of primary murine hTNFtg FLS, Rsk2-/y; chip 3. Samples were analyzed using murine MG_U430_2 arrays (Affymetrix)

hyb_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG_U430_2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C, rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 450.

data_processing: Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Grün) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 3 chips of group 1 were compared to the 3 chips of group 2 and chips within one group were compared to each other in both directions. The following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between 9 SLR values of Experiment group vs. Baseline group and 12 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls of Experiment group vs. Baseline group have to be in the same direction. Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria. These are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (in one direction only).

source_name: hTNFtg;Rsk2-/y FLS

growth_protocol: Murine fibroblast-like synoviocytes (FLS) were isolated from the hind paws of hTNFtg and hTNFtg;Rsk2-/y (background: C57BL/6) 6 - 9 week-old mice via collagenase digestion. Cells were cultured in DMEM medium supplemented with 10% heat inactivated fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml) and gentamycin (0.5 µg/ml). All cell cultures were carried out at 37 °C in 5% CO2. The cells of two mice were pooled and cultivated for at least 3 passages before usage. For characterization, the cells were tested for the surface expression of CD90.2 and VCAM1 by flow cytometric analysis (> 95%), whereas CD11b as a monocytic marker had to be absent (< 5%).

extract_protocol: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction, including the recommended DNase digest. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).

tag: genetic background

value: male

value: primary murine fibroblast-like synoviocytes (FLS)

tag: genotype

value: human TNF transgenic and knockout in Rsk2 gene (Rsk2-/y FLS)


title: Gene expression of murine fibroblast-like synoviocytes Grp2_C57BL6_2_FLS_TNFtg

description: Gene-expression profiling of primary murine hTNFtg FLS, Rsk2+/y; chip 2. Samples were analyzed using murine MG_U430_2 arrays (Affymetrix)

source_name: hTNFtg FLS

label_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG_U430_2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C, rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 450.

treatment_protocol: Murine fibroblast-like synoviocytes (FLS) were isolated from the hind paws of hTNFtg and hTNFtg;Rsk2-/y (background: C57BL/6) 6 - 9 week-old mice via collagenase digestion. Cells were cultured in DMEM medium supplemented with 10% heat inactivated fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml) and gentamycin (0.5 µg/ml). All cell cultures were carried out at 37 °C in 5% CO2. The cells of two mice were pooled and cultivated for at least 3 passages before usage. For characterization, the cells were tested for the surface expression of CD90.2 and VCAM1 by flow cytometric analysis (> 95%), whereas CD11b as a monocytic marker had to be absent (< 5%).

value: human TNF transgenic without knockout (Rsk2+/y FLS)


title: Group 5 chip 7: ND_7_CD3CD4CD45_rest

status: Public on Jun 17 2014

description:
Group 5: PMA/Iono stimulated (3h) unrelated PB memory CD4+CD69- T cells, N=8
Gene expression of human memory CD4+ T cell subsets in BM and PB of healthy donors.

hyb_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The HG-U133A plus 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

data_processing: Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. To get an overview for all differentially regulated genes each chip of the five groups was compared to each chip of any other group only in direction Experiment vs. Baseline group. Within each group all chips were compared to all others in both directions. The following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values Experiment vs. Baseline group, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between SLR values of Experiment group vs. Baseline group and SLR values within both groups (the latter giving a mean SLR value of zero); p-values had to be <0.05/(54675*(#SLRs Experiment-vs-Baseline + #SLRs Experiment-vs-Experiment + #SLRs Baseline-vs-Baseline)). As second possibility more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) e.g. for group 1 (4) vs. group 4 (8 chips) 17 or more of all 32 Change calls had to be in the same direction (increased or decreased). Significantly differentially expressed genes, were filtered using both default parameter sets (I or D) of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (one direction). To reduce the exorbitant high number of differentially regulated genes, the most homogeneous genes were selected with default parameters of pattern detection function of Genes@Work (Stolovitzky, IBM); this tool was also used for hierarchical clustering.

source_name: Group 5: PMA/Iono sti_PB_CD3CD4CD45

growth_protocol: Groups 1-3 were ex vivo memory CD4+ T-cell subsets isolated from paired BM and PB samples according to CD69 expression, without any further treatment before total RNA extraction and transcriptome analysis.  Group 4 were ex vivo memory CD4+ T cells from 8 unrelated 8 donors, without any further treatment before total RNA extraction transcriptome analysis.   Group 5 were ex vivo memory cells isolated from unrelated 8 donors (as in group 4) and stimulated with PMA (10 ng/ml) and ionomycin (1 µg/ml) for 3 h in RPMI 1640 medium supplemented with 1% glutamax, penicillin (100 U/ml), streptomycin (100 µg/ml), and 5% heat inactivated human AB serum. All cell cultures were carried out at 37 °C with 5% CO2. After stimulation cells were harvested and directly subjected to total RNA extraction and transcriptome analysis.

extract_protocol: Total RNA was extracted using a NucleoSpin RNA XS Kit (Macherey-Nagel) for samples in groups 1-3 or RNeasy Mini Kit (Qiagen) for samples in groups 4 and 5, according to the manufacturer’s instructions, including the recommended DNase digest. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step.

value: memory Th cells

value: peripheral blood (PB)

value: restimulated with PMA and iono for 3 h

value: Group 5:unrelated PB memory CD4+CD69- T cells


title: Hollow Fibre 5


title: Group 5 chip 3: ND_3_CD3CD4CD45_rest

label_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The HG-U133A plus 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.


title: Group 5 chip 6: ND_6_CD3CD4CD45_rest


title: Gene expression of murine fibroblast-like synoviocytes Grp2_C57BL6_1_FLS_TNFtg

description: Gene-expression profiling of primary murine hTNFtg FLS, Rsk2+/y; chip 1. Samples were analyzed using murine MG_U430_2 arrays (Affymetrix)


title: Gene expression of murine fibroblast-like synoviocytes Grp2_C57BL6_3_FLS_TNFtg

description: Gene-expression profiling of primary murine hTNFtg FLS, Rsk2+/y; chip 3. Samples were analyzed using murine MG_U430_2 arrays (Affymetrix)


title: Group 5 chip 8: ND_8_CD3CD4CD45_rest


title: Gene expression of murine fibroblast-like synoviocytes Grp1_C57BL6_2_FLS_TNFtg_plus_RSK2-KO

description: Gene-expression profiling of primary murine hTNFtg FLS, Rsk2-/y; chip 2. Samples were analyzed using murine MG_U430_2 arrays (Affymetrix)


title: Group 5 chip 5: ND_5_CD3CD4CD45_rest


title: Group 5 chip 1: ND_1_CD3CD4CD45_rest


title: Group 5 chip 4: ND_4_CD3CD4CD45_rest


title: Gene expression of murine fibroblast-like synoviocytes Grp1_C57BL6_1_FLS_TNFtg_plus_RSK2-KO

description: Gene-expression profiling of primary murine hTNFtg FLS, Rsk2-/y; chip 1. Samples were analyzed using murine MG_U430_2 arrays (Affymetrix)


title: Group 5 chip 2: ND_2_CD3CD4CD45_rest


title: Group 4 chip 7: ND_7_CD3CD4CD45_unst

description:
Group 4: ex vivo unrelated PB memory CD4+CD69- T cells, untreated, N=8
Gene expression of human memory CD4+ T cell subsets in BM and PB of healthy donors.

source_name: Group 4: ex vivo_PB_CD3CD4CD45

value: ex vivo, untreated

value: Group 4:unrelated PB memory CD4+CD69- T cells


title: Group 4 chip 8: ND_8_CD3CD4CD45_unst


title: Group 4 chip 4: ND_4_CD3CD4CD45_unst


title: Group 4 chip 6: ND_6_CD3CD4CD45_unst


title: Group 4 chip 3: ND_3_CD3CD4CD45_unst


title: Group 4 chip 5: ND_5_CD3CD4CD45_unst


title: Grp3_Chip2_CD44negCD127pos_spl_exvivo

description: Naive (CD44-CD127+) CD8+CD3+ T cells from murine spleen, ex vivo isolated; group 3, chip 2

value: female

value: Spleen

value: CD8+CD3+ T cells; Naive (CD44-CD127+); 3-12 mice were pooled

value: CD8+ T cells were first enriched with α-CD8 α microbeads (Miltenyi)

value: Naive (CD44-CD127+) CD8+CD3+ T cells were cytometrically sorted with FACS Aria or Influx (BD Biosciences)

value: not activated; ex vivo isolated

value: No cultivation, immediately lysed with RA-1 buffer (Macherey-Nagel) for RNA preparation


title: Grp1_Chip1_CD44posCD127pos_spl_exvivo

description: Memory (CD44+CD127+) CD8+CD3+ T cells from murine spleen, ex vivo isolated; group 1, chip 1

value: CD8+CD3+ T cells; Memory (CD44+CD127+); 3-12 mice were pooled

value: Memory (CD44+CD127+) CD8+CD3+ T cells were cytometrically sorted with FACS Aria or Influx (BD Biosciences)


title: Grp2_Chip3_CD44posCD127pos_bm_exvivo

description: Memory (CD44+CD127+) CD8+CD3+ T cells from murine bone marrow, ex vivo isolated; group 2, chip 3

value: Bone marrow


title: Group 3 chip 2: ND_5_PB-CD4_CD45Ro+CD69-

description:
Group 3: ex vivo paired PB memory CD4+CD69- T cells, untreated, N=4
Gene expression of human memory CD4+ T cell subsets in BM and PB of healthy donors.

source_name: Group 3: ex vivo_PB-CD4_CD45Ro+CD69-

value: Group 3:paired PB memory CD4+CD69- T cells


title: Group 3 chip 3: ND_6_PB-CD4_CD45Ro+CD69-

value: memory Th cells


title: Group 1 chip 4: ND_7_BM-CD4_CD45Ro+CD69+

description:
Group 1: ex vivo paired BM memory CD4+CD69+ T cells, untreated, N=4
Gene expression of human memory CD4+ T cell subsets in BM and PB of healthy donors.

source_name: Group 1: ex vivo_BM-CD4_CD45Ro+CD69+

value: bone marrow (BM)

value: Group 1:paired BM memory CD4+CD69+ T cells


title: Group 2 chip 1: ND_4_BM-CD4_CD45Ro+CD69-

description:
Group 2: ex vivo paired BM memory CD4+CD69- T cells, untreated, N=4
Gene expression of human memory CD4+ T cell subsets in BM and PB of healthy donors.

source_name: Group 2:  ex vivo_BM-CD4_CD45Ro+CD69-

value: Group 2:paired BM memory CD4+CD69- T cells


title: Group 1 chip 2: ND_5_BM-CD4_CD45Ro+CD69+

value: ex vivo, untreated


title: MF.PPAR-.Lu#4

status: Public on May 23 2014

city: Boston

first: Liang

last: Yang

state: MA

address: 77 Avenue Louis Pasteur

department: Pathology

country: USA

institute: Harvard Medical School

hyb_protocol: Affy 1.0 ST Hyb Kit

scan_protocol: Affymetrix GeneChip Scanner

data_processing: The datasets were pre-filtered to keep only those probesets for which a gene symbol could be found in the Affymetrix annotation. CEL files were normalized using Affymetrix Power Tools on the predefined probeset ID list mentioned above, and using the standard RMA workflow (background adjustment, quantile normalization, median polish probeset summarization). Output on 2^ scale.

source_name: Lung

label_protocol: Affy GeneChip WT cDNA Synthesis kit/cRNA Cleanup kit

extract_protocol: Trizol

tag: age

value: 6 weeks

tag: exclusion markers

value: MHCII- CD11b- CD103-

tag: phenotype markers

value: CD11c+ MHCII- CD11b- CD103- SiglecF+

tag: separation method

value: dissection / Trimming

tag: Sex

value: male


title: Group 2 chip 3: ND_6_BM-CD4_CD45Ro+CD69-

tag: tissue


title: Group 1 chip 1: ND_4_BM-CD4_CD45Ro+CD69+


title: Group 3 chip 1: ND_4_PB-CD4_CD45Ro+CD69-

value: peripheral blood (PB)


title: 2: IL-10-non-secreting Th1 cells with activation of Notch signaling pathway derived from in vitro polarized TH1 cells

status: Public on May 08 2014

description:
Chip2_C57Bl6_CD4CD62L_TH1notch_IL10neg_430_2
Primary naïve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions (10 ng/ml rmIL-12, 10 µg/ml anti-IL-4 (11B11). For activation, 0.25E06 naïve T cells were co-cultured with 0.75E06 MACSi Beads that were coated with anti-CD3 and anti-CD28 (30 µg of total primary IgG antibody per 1.0E08 beads) prior to seeding. Notch activation via Dll4 was induced by additional co-culture with MACSi Beads covalently coated with recombinant mouse Dll4. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to obtain IL-10-non-secreting cells.

hyb_protocol: The Mouse Genome 430 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

scan_protocol: Arrays were scanned with an Affymetrix GeneChip Scanner 3000, using the GCOS software version 1.1.1. from Affymetrix.

data_processing: Data were analyzed using an Excel spreadsheet with original GCOS CHP file Signals. Transcripts up-regulated > 2 fold in IL-10 producing vs. non-producing Th1 cells and a detection p-value < 0.05 in at least one of the chips were filtered and analysed in silico. The AmiGO web interface (Ashburner et al., 2000) was used to filter those transcripts that were associated with a Gene Ontology term including "transcription" and/or their involvement in the biological process of "positive regulation of gene expression" (GO:0010628). Further validation of all these filtered transcripts was done via qPCR.

source_name: IL-10-negative Th1 cells with activation of the Notch signaling pathway

label_protocol: Labeling of total RNA was performed using the two cycle GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturer's instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, two times amplified, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays.

extract_protocol: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instruction, including the recommended DNase digest. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip software and the amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).

treatment_protocol: Naïve T helper cells were enriched magnetically from lymph nodes and spleens of C57BL/6 wildtype mice after lysis of erythrocytes using EL buffer with the magnetic beads from the Multisort Kit from Miltenyi. Next, the enriched cells were cultured in an APC-free system under Th1 polarizing conditions in the absence or presence of a Notch inducing signal (Dll4). After 5 days of culture, cells were restimulated (PMA/Iono), and subjected to an IL-10 secretion assay. Dead cells were stained with propidiumiodide and CD4 using a fluorescently labeled antibody. These cells were then sorted by Aria or DIVA FACSorter machines. Chip 1 and 2 (with activation of the Notch signaling pathway): (1) Living CD4+ TH1 Notch IL-10 pos cells, (2) Living CD4+ TH1 Notch IL-10 neg cells. Chip 3 and 4 (without activation of the Notch signaling pathway): (3) Living CD4+ TH1 Control IL-10 pos cells, (4) Living CD4+ TH1 Control IL-10 neg cells.

tag: strain/background

tag: genotype/variation

value: wildtype

tag: cell type

value: primary naïve murine CD4+ cells

value: erythrocyte lysis (EL)

value: depletion of CD25- cells and positive enrichment of CD4+ and CD62L+ cells

value: Th1 polarizing conditions for 5 days

tag: notch signaling pathway activation

value: co-cultured with MACSi Beads covalently coated with recombinant mouse Notch ligand Delta-like 4 (Dll4)

tag: cell restimulation

value: restimulated with PMA/Ionomycin

tag: il10 staining

value: secreted IL-10 was stained using an IL-10 secretion assay (Miltenyi Biotec)

value: living CD4+ cells not secreting IL-10 (IL-10neg)


title: MF.PPAR-.Lu#3


title: Group 13 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND4  PAA 24 h 20 °C

status: Public on Mar 24 2014

description: Gene-expression profiling of peripheral human effector/memory T helper lymphocytes, enriched via CD4+ Whole Blood MACS without delay. Analyzed using human U133A plus 2.0 arrays.

data_processing: Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Grün) with the BioRetis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the BioRetis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 2 chips of each group were compared to all 2 chips of every other group and the 2 chips within one group were compared to each other in both directions. The following parameters of absolute and comparative analysis were included in the BioRetis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between 4 SLR values of Experiment group vs. Baseline group and 4 SLR values within both groups (the latter 4 giving a mean SLR value of zero; p-value had to be <= 1.143E-07) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 3 or more of all 4 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameter set" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probe sets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probe sets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (one direction).

source_name: CD3CD4CD45Ro+ Tcells PAA 24 h 20 °C

growth_protocol: Blood was drawn directly into 10 ml preservative-free EDTA Vacutainer tubes (Becton Dickinson), one full vial for each tested procedure and storage condition.   Represented in groups 1 - 8 we tested three main ways to enrich T lymphocytes before CD3+CD4+CD45RO+ fluorescence activated cell sorting (FACS) for effector/memory T helper (T E/M) lymphocytes. These included magnetic cell sorting with Whole Blood CD4 beads, density gradient centrifugation, alone or in combination with CD3 MACS and lysis of erythrocytes at 4 °C or RT followed by MACS depletion of CD15+ cells. As a high control for signs of activation T cells in group 8 were stimulated for 3 hours after FACS with Phorbol-12-myristate-13-acetate and Ionomycin (PMA/Iono).  Represented in groups 9 - 13 we tested the influence of five different storage conditions on the gene expression pattern in comparison to samples processed in the same manner without delay (group 5).  To achieve equal purity in all samples the CD3+CD4+CD45RO+ cell fraction was sorted to >98% purity using an Aria cell sorter. Sorted cells were pelleted and cell pellets were directly lysed in TRIzol (Invitrogen) by repetitive pipetting and vortexing, and incubated for 5 min at ambient temperature. Samples were than frozen at -80°C until RNA extraction.

extract_protocol: Total RNA was extracted using the miRNeasy Kit (Qiagen) according to the manufacturer’s instruction, including the recommended DNase digest. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).

treatment_protocol: Represented in groups 1-8 we tested three main ways to enrich T lymphocytes before CD3+CD4+CD45RO+ fluorescence activated cell sorting (FACS) for effector/memory T helper (TE/M) lymphocytes. These included magnetic cell sorting with Whole Blood CD4 beads, density gradient centrifugation, alone or in combination with CD3 MACS and lysis of erythrocytes at 4°C or RT followed by MACS depletion of CD15+ cells. As a high control for signs of activation T cells in group 8 were stimulated for 3 hours after FACS with Phorbol-12-myristate-13-acetate and Ionomycin (PMA/Iono).  Represented in groups 9-13 we tested the influence of five different storage conditions on the gene expression pattern in comparison to samples processed in the same manner without delay (group 5).  To achieve equal purity in all samples the CD3+CD4+CD45RO+ cell fraction was sorted to >98% purity using an Aria cell sorter. Sorted cells were pelleted and cell pellets were directly lysed in TRIzol (Invitrogen) by repetitive pipetting and vortexing, and incubated for 5 min at ambient temperature. Samples were than frozen at -80°C until used for RNA extraction

value: peripheral blood effector/memory T helper lymphocytes

tag: pre-enrichment

value: enriched via density gradient centrifugation with LSM 1077

tag: activation

value: not activated

tag: storage before pre-enrichment

value: cell isolation after storage of buffy coat for 24 h at 20 °C

tag: flow cytometric cell sorting

value: CD3+CD4+CD45Ro+ T cells


title: Group 13 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND3  PAA 24 h 20 °C


title: 3: IL-10-secreting Th1 cells without activation of Notch signaling pathway derived from in vitro polarized TH1 cells

description:
Chip3_C57Bl6_CD4CD62L_TH1control_IL10pos_430_2
Primary naïve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions (10 ng/ml rmIL-12, 10 µg/ml anti-IL-4 (11B11). For activation, 0.25E06 naïve T cells were co-cultured with 0.75E06 MACSi Beads that were coated with anti-CD3 and anti-CD28 (30 µg of total primary IgG antibody per 1.0E08 beads) prior to seeding. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to obtain IL-10-secreting cells.

source_name: IL-10-positive Th1 cells without activation of the Notch signaling pathway

tag: separation

tag: magnetic cell sorting

tag: cell cultivation

value: not co-cultured with MACSi Beads covalently coated with recombinant mouse Notch ligand Delta-like 4 (Dll4)

value: living CD4+ cells secreting IL10 (IL-10pos)


title: Group 1 chip 3: ND_6_BM-CD4_CD45Ro+CD69+

tag: cells

value: memory Th cells

value: bone marrow (BM)

value: ex vivo, untreated

value: Group 1:paired BM memory CD4+CD69+ T cells


title: MF.PPAR-.Lu#2

tag: age

value: 6 weeks

tag: exclusion markers

value: MHCII- CD11b- CD103-

tag: genetic background

tag: phenotype markers

value: CD11c+ MHCII- CD11b- CD103- SiglecF+

tag: separation method

value: dissection / Trimming

value: male


title: 1: IL-10-secreting Th1 cells with activation of Notch signaling pathway derived from in vitro polarized TH1 cells

description:
Chip1_C57Bl6_CD4CD62L_TH1notch_IL10pos_430_2
Primary naïve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions (10 ng/ml rmIL-12, 10 µg/ml anti-IL-4 (11B11). For activation, 0.25E06 naïve T cells were co-cultured with 0.75E06 MACSi Beads that were coated with anti-CD3 and anti-CD28 (30 µg of total primary IgG antibody per 1.0E08 beads) prior to seeding. Notch activation via Dll4 was induced by additional co-culture with MACSi Beads covalently coated with recombinant mouse Dll4. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to obtain IL-10-secreting cells.

source_name: IL-10-positive Th1 cells with activation of the Notch signaling pathway

tag: strain/background

tag: genotype/variation

value: wildtype

tag: cell type

value: primary naïve murine CD4+ cells

value: erythrocyte lysis (EL)

value: depletion of CD25- cells and positive enrichment of CD4+ and CD62L+ cells

value: Th1 polarizing conditions for 5 days

value: co-cultured with MACSi Beads covalently coated with recombinant mouse Notch ligand Delta-like 4 (Dll4)

value: restimulated with PMA/Ionomycin

value: secreted IL-10 was stained using an IL-10 secretion assay (Miltenyi Biotec)


title: Group 11 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 PAA 2 h 20 °C

source_name: CD3CD4CD45Ro+ Tcells PAA 2 h 20 °C

value: peripheral blood effector/memory T helper lymphocytes

value: enriched via density gradient centrifugation with LSM 1077

value: not activated

value: cell isolation after storage of whole blood for 2 h at 20 °C

value: CD3+CD4+CD45Ro+ T cells


title: Group 4 chip 1: ND_1_CD3CD4CD45_unst

value: memory Th cells

tag: tissue

value: peripheral blood (PB)

value: ex vivo, untreated

value: Group 4:unrelated PB memory CD4+CD69- T cells


title: Group 12 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 PAA 6 h 20 °C

source_name: CD3CD4CD45Ro+ Tcells PAA 6 h 20 °C

tag: pre-enrichment

value: cell isolation after storage of whole blood for 6 h at 20 °C


title: Group 10 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 PAA 6 h 4 °C

source_name: CD3CD4CD45Ro+ Tcells PAA 6 h 4 °C

tag: activation

value: cell isolation after storage of whole blood for 6 h at 4 °C


title: Group 11 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 PAA 2 h 20 °C

tag: storage before pre-enrichment


title: Group 12 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 PAA 6 h 20 °C

tag: flow cytometric cell sorting


title: Group 8 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 Ery4rest

source_name: CD3CD4CD45Ro+ Tcells Ery4rest

value: enriched via erythrocyte lysis at 4 °C

value: stimulation after FACS with PMA/iono for 3 h at 37 °C

value: cell isolation without delay


title: Group 9 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 PAA 2 h 4 °C

source_name: CD3CD4CD45Ro+ Tcells PAA 2h 4 °C

value: cell isolation after storage of whole blood for 2 h at 4 °C


title: Group 10 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 PAA 6 h 4 °C


title: Group 7 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 Ery4unst

source_name: CD3CD4CD45Ro+ Tcells Ery4unst


title: Group 5 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 PAA

source_name: CD3CD4CD45Ro+ Tcells PAA 0 h


title: Group 6 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 Ery20

source_name: CD3CD4CD45Ro+ Tcells Ery20

value: enriched via erythrocyte lysis at 20 °C


title: Group 8 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 Ery4rest


title: Group 9 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 PAA 2 h 4 °C


title: Group 6 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 Ery20


title: Group 7 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 Ery4unst


title: Group 2 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 WBB

source_name: CD3CD4CD45Ro+ Tcells WBB

value: enriched via CD4+Whole Blood MACS


title: Group 5 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 PAA

value: peripheral blood effector/memory T helper lymphocytes


title: Group 4 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 UP

source_name: CD3CD4CD45Ro+ Tcells UP

value: enriched via density gradient centrifugation with PM400


title: Group 4 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 UP

value: not activated


title: Group 3 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 UP+CD3M

source_name: CD3CD4CD45Ro+ Tcells UP+CD3M

value: enriched via density gradient centrifugation with PM400 and CD3 MACS


title: Group 1 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 WBB+AMD

source_name: CD3CD4CD45Ro+ Tcells WBB+AMD

value: enriched via CD4+ Whole Blood MACS with addition of Actinomycin D

value: CD3+CD4+CD45Ro+ T cells


title: Group 3 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 UP+CD3M


title: Group 1 chip 1: Gene expression of human CD3+CD4+CD45Ro+ cells ND5 WBB+AMD


title: Group 2 chip 2: Gene expression of human CD3+CD4+CD45Ro+ cells ND6 WBB


title: Group 2 chip 4: ND_7_BM-CD4_CD45Ro+CD69-

source_name: Group 2:  ex vivo_BM-CD4_CD45Ro+CD69-

value: memory Th cells

value: bone marrow (BM)

value: Group 2:paired BM memory CD4+CD69- T cells


title: Group 1 chip 2: Grp1_IL-10pos_B_cells_2

description: Gene-expression profiling of primary human B cells secreting IL-10 following activation with anti-CD40, IL-4 and CpG for 2 days.

hyb_protocol: The HG-U133A plus 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

data_processing: Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 2 chips of group 1 were compared to the 2 chips of group 2 and chips within one group were compared to each other in both directions. The following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between 4 SLR values of Experiment group vs. Baseline group and 4 SLR values within both groups (the latter 4 giving a mean SLR value of zero; p-value had to be <= 1.143E-07) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 3 or more of all 4 Change calls have to be in the same direction. Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (one direction).

source_name: IL-10pos B cells

label_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays.

treatment_protocol: B cells were purified magnetically from PBMCs of healthy donors using anti-CD19-coupled magnetic beads. Cells were cultured in RPMI 1640 culture medium supplemented with L-glutamine (2 mmol/L), penicillin (100 U/ml), streptomycin (100 µg/ml), 10 µmol/L mercaptoethanol and 10% heat inactivated fetal calf serum. All cell cultures were carried out at 37 °C in 5% CO2. After 48 h stimulation with 1 µg/ml anti-CD40, 10 ng IL-4 and 3 µg CpG2006, B cells were restimulated for 3 h with phorbol myristate acetate (PMA, 10 ng/ml) and ionomycin (1 µg/ml). Next, the cells were coated with a bivalent antibody matrix against CD45 and IL-10 and after 1 h, secreted IL-10 was detected with a 2nd, fluorescent IL-10 specific antibody and the cells were sorted by Aria FACSorter machine according to expression of IL-10 or not.

value: peripheral CD19pos B cells

tag: activated by

value: by CpG2006, anti-CD40 and IL-4 for 2 days

value: IL-10pos B cells


title: Group 1 chip 1: Grp1_IL-10pos_B_cells_1


title: Group 2 chip 1: Grp2_IL-10neg_B_cells_1

description: Gene-expression profiling of primary human B cells not secreting IL-10 following activation with anti-CD40, IL-4 and CpG for 2 days.

source_name: IL-10neg B cells

value: IL-10neg B cells


title: Differentially activated CD8 T cells in liver and gut: Naïve CD8 OT-I T cells (naïve) Chip 2

status: Public on Feb 25 2014

description: Gene expression profiles of (a) naïve CD8 OT-I T cells, (b) Liver-activated CD8 OT-I T cells, and (c) gut-activated CD8 OT-I T cells prepared from lymph nodes and spleens of OT-I mice, the livers of TF-OVA mice and mesenteric lymph nodes of iFABP-OVA mice were compared using Affymetrix MG 430 2.0 GeneChip arrays. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized in duplicates for each of the three groups to the GeneChip arrays. Group 1: naïve OT-I T cells, Group 2: liver-activated OT-I T cells, Group 3: gut-activated OT-I T cells.

hyb_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 300 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to the GeneChip arrays. The MG 430 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 450.

scan_protocol: Arrays were scanned with an Affymetrix GeneChip Scanner 3000

data_processing: Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All two chips of one group were compared to any of the two chips of the second group (4 comparisons between two groups) and all chips were additionally compared within each group, (2 comparisons in both directions within each group). The following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal Increase, MI; Decrease, D; Marginal Decrease, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – four different t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using (a) strict Bonferroni corrected Welch t tests between 4 SLR values of group 1 vs. group 2 and 2 plus 2 SLR values within both groups (the latter 4 giving a mean SLR value of zero; p-value had to be <= 1.38578E-07) or (b) more than 50% of non parametrically calculated Change calls (call after calculation of p value with Mann-Whitney U test, GCOS) i.e. 3 or more of all 4 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using the both default parameter sets of filter criteria (for up- and down regulated genes separately); these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips, validated with the Affymetrix Latin Square dataset (accessible via public login without registration in the public data sets SGU of Bioretis) and compared successfully to Significance Analysis of Microarrays (SAM) and dChip in Menssen et al., 2009. Advantage of High Performance Chip Data Analysis (HPCDA) is amongst others the possibility to use always the same (default) parameters for all different datasets resulting in approx. 65 (Latin Square data set) up to 10,000 significant differentially regulated genes without parameter adjusting by the user.

source_name: CD8 OT-I T-cell subsets

growth_protocol: Naïve CD8 OT-I T cells were isolated from lymph nodes and spleens of OT-I mice using the isolation kits from Miltenyi Biotec. CD8 OT-I T cells activated by liver-derived and small intestine-derived antigen, positive for CFSE were sorted from livers of TF-OVA mice or from mesenteric lymph nodes of iFABP-OVA mice using a FACSAria (BD Biosciences).

extract_protocol: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and amount with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).

tag: strain

value: OT-I, TF-OVA, iFABP-OVA mice

tag: gender

value: male

value: Lymph node and spleen, liver, mesenteric lymph node


title: Differentially activated CD8 T cells in liver and gut: Gut-activated CD8 OT-I T cells (mes) Chip 2

label_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 300 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to the GeneChip arrays. The MG 430 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 450.

treatment_protocol: Naïve CD8 OT-I T cells were isolated from lymph nodes and spleens of OT-I mice using the isolation kits from Miltenyi Biotec. CD8 OT-I T cells activated by liver-derived and small intestine-derived antigen, positive for CFSE were sorted from livers of TF-OVA mice or from mesenteric lymph nodes of iFABP-OVA mice using a FACSAria (BD Biosciences).


title: Differentially activated CD8 T cells in liver and gut: Gut-activated CD8 OT-I T cells (mes) Chip 1


title: Group5-7 LPS + anti-CD40 activated murine B cells 72h

status: Public on Feb 24 2014

description: Gene expression profiles of naïve, LPS or LPS + anti-CD40 activated B cells prepared from the spleens of C57BL/6 mice were compared using Affymetrix MG430Av2 GeneChip arrays. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized in quadruplicates for each of the five groups to the GeneChip arrays. Twenty chips in these five groups were hybridized and analyzed: Group1 naïve B cells, Group2 LPS activated B cells 24h, Group3 LPS + anti-CD40 activated B cells 24h, Group4 LPS activated B cells 72h, and Group5 LPS + anti-CD40 activated B cells 72h.

hyb_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG430Av2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

scan_protocol: Arrays were scanned with an Affymetrix GeneChip Scanner 3000, using the GCOS software from Affymetrix.

data_processing:
In DRFZ with GeneChip Operating System (GCOS, Affymetrix) generated CHP and CEL files were analyzed at INRA, France (Luc Jouneau and Pierre Boudinot). Only chips, which fulfilled all chip and hybridization quality criteria, were selected for analysis. Normalization has been performed using R package GCRMA. To detect which probe sets were expressed in a given sample and to get Present/Marginal/Absent calls mas5calls function of affy R package was used. Differential expression was detected using limma R package. Some of the results were compared to High Performance Chip Data Analysis (HPCDA) in DRFZ, using the BioRetis database (Joachim R. Grün and Andreas Grützkau).
All chips were imported into BioRetis (http://www.bioretis-analysis.de) as publicly available datasets and comparisons of all groups to all other groups were calculated. If you want to obtain one or more lists of HPCDA significant genes you should register to get an BioRetis account and search for those here listed SPL datasets you are interested in (4302 is the mouse chip type, used):
SPL_770: C57BL6_B-cells_LPS_24h_vs_C57BL6_B-cells_naive_0h_4302,
SPL_771: C57BL6_B-cells_LPS+CD40_24h_vs_C57BL6_B-cells_naive_0h_4302,
SPL_772: C57BL6_B-cells_LPS_72h_vs_C57BL6_B-cells_naive_0h_4302,
SPL_773: C57BL6_B-cells_LPS+CD40_72h_vs_C57BL6_B-cells_naive_0h_4302,
SPL_774: C57BL6_B-cells_LPS+CD40_24h_vs_C57BL6_B-cells_LPS_24h_4302,
SPL_775: C57BL6_B-cells_LPS_72h_vs_C57BL6_B-cells_LPS_24h_4302,
SPL_776: C57BL6_B-cells_LPS+CD40_72h_vs_C57BL6_B-cells_LPS_24h_4302,
SPL_777: C57BL6_B-cells_LPS_72h_vs_C57BL6_B-cells_LPS+CD40_24h_4302,
SPL_778: C57BL6_B-cells_LPS+CD40_72h_vs_C57BL6_B-cells_LPS+CD40_24h_4302,
SPL_779: C57BL6_B-cells_LPS+CD40_72h_vs_C57BL6_B-cells_LPS_72h_4302.
To see already published (SPL) datasets and to get access to the default query parameters of BioRetis DB you have to check the four checkboxes that will be found on page Account/Profile (and save it). Please use the default query parameters for Increased or Decreased genes one after the other. Click on Comparison/View single results, select one of the above listed SPL analyses and click on "Next". Click on "Chose an existing parameterset" and select "default increased in EG” for increased in experiment group or "default decreased in EG” for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of HPCDA significantly changed increased or decreased probesets, respectively.

source_name: LPS + anti-CD40 72h activated splenic B cells

growth_protocol: Splenic B cells CD19+ were purified by MACS (Miltenyi) using CD43, CD11c, CD11b, DX5 and CD90 microbeads, which resulted in high purity (>99 %). B cells were activated in RPMI medium (Gibco) supplemented with 10 % FCS (Gibco) and 1 % Penicillin/Streptomycin (PAA Laboratories), with LPS (O55:B5, 1 µg/ml) or with LPS + anti-CD40 (FGK45, 10 µg/ml) for 24h and 72h.

value: Spleen

tag: cells

value: B-cells, LPS + anti-CD40 72h activated


title: Differentially activated CD8 T cells in liver and gut: Naïve CD8 OT-I T cells (naïve) Chip 1


title: Differentially activated CD8 T cells in liver and gut: Liver-activated CD8 OT-I T cells (liver) Chip 1


title: Group 2 chip 2: Grp2_IL-10neg_B_cells_2


title: Group5-44 LPS + anti-CD40 activated murine B cells 72h

label_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays. The MG430Av2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.

treatment_protocol: Splenic B cells CD19+ were purified by MACS (Miltenyi) using CD43, CD11c, CD11b, DX5 and CD90 microbeads, which resulted in high purity (>99 %). B cells were activated in RPMI medium (Gibco) supplemented with 10 % FCS (Gibco) and 1 % Penicillin/Streptomycin (PAA Laboratories), with LPS (O55:B5, 1 µg/ml) or with LPS + anti-CD40 (FGK45, 10 µg/ml) for 24h and 72h.


title: Group5-45 LPS + anti-CD40 activated murine B cells 72h


title: Group5-6A LPS + anti-CD40 activated murine B cells 72h


title: Group3-5 LPS + anti-CD40 activated murine B cells 24h

source_name: LPS + anti-CD40 24h activated splenic B cells

value: B-cells, LPS + anti-CD40 24h activated


title: Group4-3 LPS activated murine B cells 72h

source_name: LPS 72h activated splenic B cells

value: B-cells, LPS 72h activated


title: Group4-6 LPS activated murine B cells 72h


title: Group2-6 LPS activated murine B cells 24h

source_name: LPS 24h activated splenic B cells

value: B-cells, 24h LPS activated


title: Group4-2 LPS activated murine B cells 72h


title: Group3-4 LPS + anti-CD40 activated murine B cells 24h


title: Group2-2 LPS activated murine B cells 24h


title: Group4-1 LPS activated murine B cells 72h


title: Group3-7 LPS + anti-CD40 activated murine B cells 24h


title: Group3-6 LPS + anti-CD40 activated murine B cells 24h


title: Differentially activated CD8 T cells in liver and gut: Liver-activated CD8 OT-I T cells (liver) Chip 2


title: Group1-3 naive murine B cells

source_name: Naïve splenic B cells

value: B-cells, naive


title: Group2-1 LPS activated murine B cells 24h


title: Group2-3 LPS activated murine B cells 24h


title: Group 2 chip 2: ND_5_BM-CD4_CD45Ro+CD69-

value: ex vivo, untreated


title: 4: IL-10-non-secreting Th1 cells without activation of Notch signaling pathway derived from in vitro polarized TH1 cells

description:
Chip4_C57Bl6_CD4CD62L_TH1control_IL10neg_430_2
Primary naïve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions (10 ng/ml rmIL-12, 10 µg/ml anti-IL-4 (11B11). For activation, 0.25E06 naïve T cells were co-cultured with 0.75E06 MACSi Beads that were coated with anti-CD3 and anti-CD28 (30 µg of total primary IgG antibody per 1.0E08 beads) prior to seeding. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to obtain IL-10-non-secreting cells.

source_name: IL-10-negative Th1 cells without activation of the Notch signaling pathway

value: wildtype

value: primary naïve murine CD4+ cells

tag: separation

value: erythrocyte lysis (EL)

tag: magnetic cell sorting

value: depletion of CD25- cells and positive enrichment of CD4+ and CD62L+ cells

tag: cell cultivation

value: Th1 polarizing conditions for 5 days

tag: notch signaling pathway activation

value: not co-cultured with MACSi Beads covalently coated with recombinant mouse Notch ligand Delta-like 4 (Dll4)

tag: cell restimulation

value: restimulated with PMA/Ionomycin

tag: il10 staining

value: secreted IL-10 was stained using an IL-10 secretion assay (Miltenyi Biotec)

value: living CD4+ cells not secreting IL-10 (IL-10neg)


title: Group 3 chip 4: ND_7_PB-CD4_CD45Ro+CD69-

source_name: Group 3: ex vivo_PB-CD4_CD45Ro+CD69-

value: memory Th cells

value: peripheral blood (PB)

value: Group 3:paired PB memory CD4+CD69- T cells


title: Group1-7 naive murine B cells

value: male

value: Spleen


title: Group1-6 naive murine B cells


title: Group 4 chip 2: ND_2_CD3CD4CD45_unst

source_name: Group 4: ex vivo_PB_CD3CD4CD45

value: Group 4:unrelated PB memory CD4+CD69- T cells


title: Sample 18

status: Public on Jan 21 2014

city: New York

first: Mayte

last: Suarez-Farinas

state: NY

address: 1425 Madison Ave, L2-70C, Box 1077,

institute: Mount SinaiSchool of Medicine

description: skin from normal mouse

hyb_protocol: RNA was hybridized mouse4302 chips to measure relative gene expression.

scan_protocol: Hewlett-Packard GeneArray Scanner

data_processing: GCRMA (using gcrma package from R/Bioconductor)

label: biotin

source_name: skin from normal mouse

label_protocol: GeneChip Affymetrix protocol

extract_protocol: Total RNA was extracted using RNeasy Micro Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol with on-column DNase digestion

tag: treatment

value: naïve

tag: condition

value: Normal


title: Sample 13

description: skin from IL23 mouse treated with Saline

value: saline

value: IL23 induced phenotype


title: Sample 15

source_name: skin from IL23 mouse treated with Saline


title: Sample 11


title: Sample 20

description: skin from normal mouse


title: Sample 12


title: Sample 17

description: skin from normal mouse


title: Sample 16

description: skin from normal mouse


title: Group1-2 naive murine B cells

tag: tissue

value: B-cells, naive


title: Sample 14


title: Sample 19

description: skin from normal mouse


title: ChorionicVilli_Control_3rd_10

status: Public on Jan 15 2014

data_processing: GenomeStudio 2011.1 software and R version 2.14.0

source_name: placental chorionic villus

label_protocol: Illumina HumanMethylation450 BeadChip Protocol

extract_protocol: Genomic DNA was purified using the Dneasy Blood and Tissue kit (Qiagen) followed by bisufite conversion using the EZ DNA methylation kit (Zymo) following manufacturer's protocols.

tag: gestational age (weeks)

tag: Sex

value: M

tag: genotype

value: control

value: placental chorionic villus


title: ChorionicVilli_Control_3rd_3


title: ChorionicVilli_Control_3rd_8


title: ChorionicVilli_Control_3rd_1

value: F


title: ChorionicVilli_Control_3rd_6


title: ChorionicVilli_Control_3rd_7


title: ChorionicVilli_Control_1st_1


title: ChorionicVilli_T16_1st_2

value: trisomy 16


title: ChorionicVilli_CPM16_3rd_9

value: confined placental mosaicism


title: Yellow fever vaccinated NDs; Group 9: CD16pos, resident monocytes; ND 55 seven days after YFV

status: Public on Jan 06 2014

description: Purified CD16-positive monocytes isolated from healthy individual ND55, 7 days after yellow fever vaccination

hyb_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted and used as template for in vitro-transcription to generate biotinylated cRNA. cRNA was fragmented and 15 µg cRNA hybridized to each of the GeneChip arrays. The HG-U133 Plus 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 450.

data_processing: Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Grün) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All chips of one SLE group were compared to each of any other ND group, and the following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between SLR values of Experiment group vs. Baseline group and SLR values within both groups (the latter always giving a mean SLR value of zero) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) have to be in the same direction. Compared were Grp1 vs. Grp4 (24 comparisons, that means at least 13 Change Calls have to be in increased or decreased direction and/or p-value < 1.3856E-08), Grp1 vs. Grp5 (24 comparisons, p-value < 1.386E-08), Grp2 vs. Grp6 (16 comparisons, p-value < 2.286E-08), Grp2 vs. Grp7 (16 comparisons, p-value < 2.286E-08), Grp3 vs. Grp8 (12 comparisons, p-value < 3.048E-08) and Grp3 vs. Grp9 (12 comparisons, p-value < 3.048E-08). Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (one direction).

source_name: CD16pos, resident monocytes from whole blood

extract_protocol: Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction, including the recommended DNase digestion. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).

treatment_protocol: A total of 50 ml peripheral blood was collected in Vacutainer heparin tubes (Becton-Dickinson, Heidelberg, Germany), and erythrocytes were lysed in EL buffer (Qiagen, Hilden, Germany). Subsequently, granulocytes were depleted using CD15-conjugated microbeads (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). The CD15-depleted fraction was stained with a CD14-fluorescein isothiocyanate (FITC) antibody (Becton-Dickinson), a CD16-APC-Cy7 antibody (Becton-Dickinson), a CD3-Vioblue antibody (Becton-Dickinson) and a CD4-FITC antibody (Becton-Dickinson). Using a FACSAria cell sorter (Becton-Dickinson), CD4pos T cells, CD16neg monocytes and CD16pos monocytes were isolated with purities and viabilities of >97%. After sorting, the cells were immediately lysed with RLT buffer (Qiagen) and frozen at -70 °C.

tag: blood donor

value: Healthy individual (ND)

tag: vaccination

value: 7 days after Yellow Fever vaccination

tag: cell type

value: CD16 positive

tag: flow cytometric cell sorting


title: ChorionicVilli_T16_1st_5


title: ChorionicVilli_T16_1st_4


title: ChorionicVilli_Control_3rd_4


title: ChorionicVilli_Control_1st_2


title: Yellow fever vaccinated NDs; Group 9: CD16pos, resident monocytes; ND 57 seven days after YFV

description: Purified CD16-positive monocytes isolated from healthy individual ND57, 7 days after yellow fever vaccination

label_protocol: Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted and used as template for in vitro-transcription to generate biotinylated cRNA. cRNA was fragmented and 15 µg cRNA hybridized to each of the GeneChip arrays. The HG-U133 Plus 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 450.


title: ChorionicVilli_Control_3rd_5


title: Yellow fever vaccinated NDs; Group 8: CD16pos, resident monocytes; ND 56 before YFV

description: Purified CD16-positive monocytes isolated from healthy individual ND56 before yellow fever vaccination


title: ChorionicVilli_Control_3rd_2


title: ChorionicVilli_Control_1st_5


title: Yellow fever vaccinated NDs; Group 8: CD16pos, resident monocytes; ND 55 before YFV

description: Purified CD16-positive monocytes isolated from healthy individual ND55 before yellow fever vaccination


title: ChorionicVilli_CPM16_3rd_7


title: ChorionicVilli_CPM16_3rd_6


title: ChorionicVilli_CPM16_3rd_1

description: EOPET


title: ChorionicVilli_T16_1st_1


title: ChorionicVilli_T16_1st_3


title: ChorionicVilli_Control_3rd_9


title: Yellow fever vaccinated NDs; Group 8: CD16pos, resident monocytes; ND 57 before YFV

description: Purified CD16-positive monocytes isolated from healthy individual ND57 before yellow fever vaccination


title: Yellow fever vaccinated NDs; Group 9: CD16pos, resident monocytes; ND 56 seven days after YFV

description: Purified CD16-positive monocytes isolated from healthy individual ND56, 7 days after yellow fever vaccination


title: ChorionicVilli_CPM16_3rd_8


title: Yellow fever vaccinated NDs; Group 7: CD16neg, inflammatory monocytes; ND 57 seven days after YFV

description: Purified CD16-negative monocytes isolated from healthy individual ND57, 7 days after yellow fever vaccination

source_name: CD16neg, inflammatory monocytes from whole blood

value: CD16 negative


title: Yellow fever vaccinated NDs; Group 7: CD16neg, inflammatory monocytes; ND 55 seven days after YFV

description: Purified CD16-negative monocytes isolated from healthy individual ND55, 7 days after yellow fever vaccination


title: ChorionicVilli_CPM16_3rd_2

description: EOPET


title: Yellow fever vaccinated NDs; Group 6: CD16neg, inflammatory monocytes; ND 56 before YFV

description: Purified CD16-negative monocytes isolated from healthy individual ND56 before yellow fever vaccination


title: ChorionicVilli_Control_1st_3


title: Yellow fever vaccinated NDs; Group 6: CD16neg, inflammatory monocytes; ND 55 before YFV

description: Purified CD16-negative monocytes isolated from healthy individual ND55 before yellow fever vaccination


title: Yellow fever vaccinated NDs; Group 6: CD16neg, inflammatory monocytes; ND 57 before YFV

description: Purified CD16-negative monocytes isolated from healthy individual ND57 before yellow fever vaccination


title: ChorionicVilli_CPM16_3rd_5

description: EOPET


title: Yellow fever vaccinated NDs; Group 5: CD4 T lymphocytes; ND 57 seven days after YFV

description: Purified CD4 T lymphocytes isolated from healthy individual ND57, 7 days after yellow fever vaccination

source_name: CD4 T lymphocytes from whole blood


title: Yellow fever vaccinated NDs; Group 7: CD16neg, inflammatory monocytes; ND 56 seven days after YFV

description: Purified CD16-negative monocytes isolated from healthy individual ND56, 7 days after yellow fever vaccination


title: ChorionicVilli_CPM16_3rd_4

description: EOPET


title: Yellow fever vaccinated NDs; Group 6: CD16neg, inflammatory monocytes; ND 54 before YFV

description: Purified CD16-negative monocytes isolated from healthy individual ND54 before yellow fever vaccination


title: Yellow fever vaccinated NDs; Group 5: CD4 T lymphocytes; ND 55 seven days after YFV

description: Purified CD4 T lymphocytes isolated from healthy individual ND55, 7 days after yellow fever vaccination


title: Yellow fever vaccinated NDs; Group 5: CD4 T lymphocytes; ND 56 seven days after YFV

description: Purified CD4 T lymphocytes isolated from healthy individual ND56, 7 days after yellow fever vaccination


title: ChorionicVilli_CPM16_3rd_3

description: EOPET


title: SLE-patients; Group 2: CD16neg, inflammatory monocytes; active SLE 1

description: Purified CD16-negative monocytes isolated from active SLE-patient 1

value: SLE patient


title: SLE-patients; Group 1: CD4 T lymphocytes; active SLE 12

description: Purified CD4 T lymphocytes isolated from active SLE-patient 12


title: SLE-patients; Group 1: CD4 T lymphocytes; active SLE 9

description: Purified CD4 T lymphocytes isolated from active SLE-patient 9


title: SLE-patients; Group 2: CD16neg, inflammatory monocytes; active SLE 2

description: Purified CD16-negative monocytes isolated from active SLE-patient 2


title: Yellow fever vaccinated NDs; Group 4: CD4 T lymphocytes; ND 56 before YFV

description: Purified CD4 T lymphocytes isolated from healthy individual ND56 before yellow fever vaccination


title: ChorionicVilli_Control_1st_4


title: Yellow fever vaccinated NDs; Group 4: CD4 T lymphocytes; ND 55 before YFV

description: Purified CD4 T lymphocytes isolated from healthy individual ND55 before yellow fever vaccination


title: SLE-patients; Group 1: CD4 T lymphocytes; active SLE 8

description: Purified CD4 T lymphocytes isolated from active SLE-patient 8


title: SLE-patients; Group 3: CD16pos, resident monocytes; active SLE 4

description: Purified CD16-positive monocytes isolated from active SLE-patient 4


title: SLE-patients; Group 2: CD16neg, inflammatory monocytes; active SLE 4

description: Purified CD16-negative monocytes isolated from active SLE-patient 4


title: SLE-patients; Group 2: CD16neg, inflammatory monocytes; active SLE 3

description: Purified CD16-negative monocytes isolated from active SLE-patient 3


title: SLE-patients; Group 3: CD16pos, resident monocytes; active SLE 2

description: Purified CD16-positive monocytes isolated from active SLE-patient 2


title: SLE-patients; Group 1: CD4 T lymphocytes; active SLE 2

description: Purified CD4 T lymphocytes isolated from active SLE-patient 2


title: Yellow fever vaccinated NDs; Group 4: CD4 T lymphocytes; ND 54 before YFV

description: Purified CD4 T lymphocytes isolated from healthy individual ND54 before yellow fever vaccination


title: SLE-patients; Group 3: CD16pos, resident monocytes; active SLE 1

description: Purified CD16-positive monocytes isolated from active SLE-patient 1


title: SLE-patients; Group 3: CD16pos, resident monocytes; active SLE 3

description: Purified CD16-positive monocytes isolated from active SLE-patient 3


title: Yellow fever vaccinated NDs; Group 5: CD4 T lymphocytes; ND 54 seven days after YFV

description: Purified CD4 T lymphocytes isolated from healthy individual ND54, 7 days after yellow fever vaccination


title: SLE-patients; Group 1: CD4 T lymphocytes; active SLE 4

description: Purified CD4 T lymphocytes isolated from active SLE-patient 4