Au-PEG_20 nanometer_15ug/mL_r2
Homo sapiens
Protocols: The undifferentiated monocytic THP.1 cell line was purchased from ATCC® (#TIB202). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.05 mM β-mercaptoethanol. The cell density was strictly kept between 0.1 to 2 million cells/mL. Undifferentiated THP.1 cells (1 milion cells per condition) were exposed to the 6 Au NPs (two diameters 5 and 20 nm, and three functional surface groups -NR3+, -COOH and -PEG) at different doses for 6 h followed by RNA extraction. Based on the preliminary cytotoxicity screening we have determined the doses inducing 10% of cell death for Au-5-NH2 (EC10= 27 µg/mL) and Au-20-NH2 (EC10= 4 µg/mL). The EC10 values for the 4 other NPs were not determined (EC10> 100 µg/mL). In this pilot study, cells were exposed: (1) to the three Au-5(-NH2/-COOH/-PEG) NPs at a concentration of 27 µg/mL (corresponding to the individual EC10 dose for the set of 5 nm size NPs), (2) to the three Au-20(-NH2/-COOH/-PEG) NPs at a concentration of 4 µg/mL (corresponding to the individual EC10 dose for the set of 20 nm size NPs) or (3) to all the Au-5 and Au-20 (-NH2/-COOH/-PEG) at a concentration of 15 µg/mL (corresponding to the average EC10 dose). The control used was cells incubated with culture media only. Each condition was performed in triplicate. After exposure, the cells were washed twice with ice-cold PBS by centrifugation 300 g, 5 min at 4°C. The cells were lysed with 350 µL of Buffer RLT Plus, supplemented with β-mercaptoethanol, from the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). Samples were stored at -80°C before RNA/DNA extraction. Qiagen protocol was strictly applied. At the end of the procedure, RNA was eluted at once with 30 µL of RNAse free water. Aliquots of 3 µL of each RNA samples were prepared for quality analysis. The concentrations of total RNA were measured with Nanodrop spectrophotometer and Qbit fluorescence. The RNA samples were carefully labelled and stored at -80°C prior to sequencing. Bulk-RNA transcriptome analysis was performed by the STRT RNA-seq method (PMID: 22481528) with minor modifications (PMID: 26874359). Briefly, ten nanogram of high-quality input RNA was converted to cDNA and amplified to form an Illumina-compatible 46-plex library. ERCC RNA Spike-In Mix 1 (cat. 4456740; ThermoScientific) was used in 1000X dilution. In total, 25 PCR cycles were used, but as six base-pair unique molecular identifiers (UMIs) were applied, only the absolute number of unique reads was calculated per analyzed sample.
ENA-FIRST-PUBLIC
2019-06-04T17:04:02Z
ENA-LAST-UPDATE
2018-08-06T09:51:00Z
External Id
SAMEA4819546
INSDC center name
Division of Chemical Biology, Department of Biology and Biological Engineering
INSDC first public
2019-06-04T17:04:02Z
INSDC last update
2018-08-06T09:51:00Z
INSDC status
public
Submitter Id
E-MTAB-7093:Au-PEG_20 nanometer_15ug/mL_r2
broker name
ArrayExpress
cell line
THP-1
cell type
monocyte
common name
human
disease
acute monocytic leukemia
organism part
blood
sample name
E-MTAB-7093:Au-PEG_20 nanometer_15ug/mL_r2
scientific_name
Homo sapiens