2020-12-01
ENA first public
2020-06-18
ENA last update
ERC000011
ENA-CHECKLIST
SAMEA6979741
External Id
Umea university
INSDC center alias
Umea university
INSDC center name
2020-12-01T04:08:12Z
INSDC first public
2020-06-18T10:58:51Z
INSDC last update
public
INSDC status
ERS4689579
SRA accession
E-MTAB-9105_2:131_G20
Submitter Id
6 to 10
week
age
ArrayExpress
http://www.ebi.ac.uk/efo/EFO_0009737
broker name
dendritic cell
http://purl.obolibrary.org/obo/CL_0000451
cell type
house mouse
common name
Protocols: Post infection, cells were treated with Fc-block (anti-mouse CD16/CD32 antibody, 1:200; BD 553142) for 15 minutes, washed with D-PBS and further incubated with a monoclonal antibody against CD11c conjugated with PE-Cyanine7 (1:200; Thermofisher 25-0114-82) for 30 minutes, washed with D-PBS and resuspended in ice-cold D-PBS. Cells were kept on ice prior to sorting. The sorting was performed with a MoFlo Astrios EQ (Beckman Coulter, USA) cell sorter using a 488 and 532 nm laser for excitation, 100 µm nozzle, sheath pressure of 25 psi and 0.1 µm sterile filtered 1 x PBS as sheath fluid. Flow sorting data was interpreted and displayed using the associated software Summit v 6.3.1. Primary bone marrow-derived dendritic cells (BMDCs) were generated as previously described (Fuks et. al., 2012, Kanatani et. al., 2017). Briefly, cells from bone marrow of 6-10 week old C57BL/6 mice (Charles River) were cultured in RPMI 1640 (Gibco) with 10% fetal bovine serum (FBS, Sigma), gentamicin (20 μg/ml; Gibco), glutamine (2 mM; Gibco) and HEPES (0.01 M; Gibco), referred to as complete medium, and supplemented with 10 ng/ml recombinant mouse GM-CSF (Peprotech). Medium was replenished on days 2,4 and 6. Loosely adherent cells were harvested on day 7/8 for experiments. The parasite lines used include GFP-expressing RH-LDMluc (type I, cloned from RH-GFPS65T) and GFP-expressing PTGluc (type II, cloned from ME49/PTG-GFPS65T). Tachyzoites were maintained by serial 2-day passaging in human foreskin fibroblast (HFF-1 SCRC-1041, American Type Culture Collection) monolayers cultured in Dulbecco‘s modified Eagle’s medium (DMEM; Gibco) with 10% FBS, gentamicin (20μg/ml), glutamine (2mM), and HEPES (0.01 M). Infection challenges, lysates and LPS On day seven or eight, DCs in complete media were challenged with freshly egressed T. gondii tachyzoites for 3 hours at MOI (multiplicity of infection) 3, or 12 hours at MOI 1.5, or treated with parasite lysates or 100ng/ml LPS for 3 hours. The parasite lysates were produced by ultrasonication of live tachyzoites, where ultrasonication was applied 3 times at 10 second intervals with 1-minute intervals on ice. CD11c staining of DCs Post infection, cells were treated with Fc-block (anti-mouse CD16/CD32 antibody, 1:200; BD 553142) for 15 minutes, washed with D-PBS and further incubated with a monoclonal antibody against CD11c conjugated with PE-Cyanine7 (1:200; Thermofisher 25-0114-82) for 30 minutes, washed with D-PBS and resuspended in ice-cold D-PBS. Cells were kept on ice prior to sorting. Individual DCs or T. gondii infected DCs were deposited into 384-well plates (Eppendorf twin.tecTM PCR plates) containing 2.3 µl lysis buffer3 using a CyCloneTM robotic arm and the most stringent single cell sort settings (single mode, 0.5 drop envelope). Side scatter was used as trigger channel and sort regions were based on Toxoplasma cells expressing GFP and the surface antibody CD11C-PE-Cy7 bound to human dendritic cells. GFP was excited at 488 nm and detected with a 530/40 nm bandpass filter whereas PE-Cy7 was excited at 532 nm and detected using a 695/70 nm bandpass filter. The plate and sample holder was kept at 4 ºC at all times during the sort. After the sort, the plates were immediately spun down and put on dry ice. Single dendritic cells or single T. gondii infected dendritic cells were sorted directly into lysis buffer and cDNA libraries were generated by the Eukaryotic Single Cell Genomics Facility at SciLifeLab, Stockholm using Smart-seq2. Smart-seq2
description
adult
developmental stage
wild type genotype
http://www.ebi.ac.uk/efo/EFO_0005168
genotype
Mus musculus
http://purl.obolibrary.org/obo/NCBITaxon_10090
organism
Mus musculus
http://purl.obolibrary.org/obo/NCBITaxon_10090
organism
bone marrow
organism part
131
plate number
G20
plate well
female
http://purl.obolibrary.org/obo/PATO_0000383
sex
C57BL/6
strain
131_G20
title