GSM4404010: Microglia from Toxoplasma gondii-infected mice 2; Mus musculus; RNA-Seq
Immediately after sacrifice mice were perfused with 30 mL of cold 1X PBS. Brains were harvested and put into cold complete RPMI media. For serum, blood from the heart was collected and allowed to clot at 4°C overnight. Brains were minced and homogenized, digested with 0.227 mg/mL collagenase/dispase and 50 U/mL DNase (Roche) at 37°C for 45 minutes. Homogenate was passed through a 70 µm filter (Corning) and washed with cRPMI. Filtered brain homogenate was then resuspended with 20 mL of 40% Percoll and spun at 650 x g for 25 minutes. Myelin was aspirated, samples were washed with cRPMI, and then resuspended in cRPMI. Single cell suspensions from tissue samples were plated in a 96-well U-bottom plate. Cells were initially incubated with 50 µL Fc block (1 µg/mL 2.4G2 Ab (BioXCell), 0.1% rat g globulin) for 10 min at room temperature. Cells were then surface stained with antibodies and a Live/Dead stain for 30 minutes at 4°C. Cells were then washed with FACS buffer (0.2% BSA and 2 mM EDTA in 1X PBS) and fixed at 4°C for 30 minutes with either 2% paraformaldehyde (PFA) or a fixation/permeabilization kit (eBioscience). Cells were then permeabilized and stained with any intracellular markers for 30 minutes at 4°C. Samples were then washed, resuspended in FACS buffer, and run on a Gallios flow cytometry (Beckman Coulter). Analysis was done using FlowJo software v.10. Antibody clones used include: CD31 (390), CD45 (30-F11), MHC-II (M5/114.15.2), NK1.1 (PK136), CD19 (1D3), CD3 (17A2), CD4 (GK1.5), CD11c (N418), CD11b (M1/70), Foxp3 (FJK-16s), Ly6G (1A8), Ly6C (HK1.4), CD8a (53-6.7), IFN-g (XMG1.2), NOS2 (CXNFT), IL-1a (ALF-161), and pro-IL-1b (NJTEN3). For cell sorting, CX3CR1creERT2 x ZsGreen 492 fl/stop/fl mice were used. After surface staining, live cells were analyzed on a BD Aria in the UVA flow cytometry core facility. Cells were sorted based on ZsGreen expression into Trizol for RNA-sequencing. RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM4404010