GSM8723638: Pooled lung resident T cells from Young_A20, YoungD1cA20, and Aged mice (Gene_expression); Mus musculus; RNA-Seq
Prior to sacrifice animals were anesthetized and administerd 3ug of fluorescently labeled anti-mouse CD45 (clone 30-F11, BD563890) in 150 uL PBS. After 3 minutes of antibody circulation mice were euthanized with isofluorane. After cardiac perfusion with PBS, mouse lungs were tracheally perfused with a digestion cocktail of Liberase TM (40 ug/mL, Millipore Sigma 5401119001) and Dnase (50 U/ml, Sigma DN25) in HBSS (+Ca+Mg) and removed from the chest. The lung was incubated in digestion cocktail for 45 mins at 37C with continuous shaking. The mixture was then washed with FACS buffer (2% FBS and 1% Penicillin-Streptomycin in DMEM) and passed through a 70 μm cell strainer. Cells were pelleted at 550xg for 5 minutes and then resuspended in red blood cell (RBC) lysis buffer for 2 minutes. After RBC lysis cells were passed through a 40 μm cell strainer. Cell suspensions were blocked with mouse FcR blocking reagent (Miltenyl Biotec 130-092-575) for 15 minutes and then incubated with the appropriate fluorescently-conjugated antibodies and Biolegend barcode antibodies in sorting buffer for 30 min at 4C. After washing with FACS buffer cells were sorted. Doublets and dead cells were excluded based on forward and side scatter and DRAQ7 (CST #7406), respectively. The following antibodies were used for staining: anti-CD45 UVC (BD 564279) , anti-CD11b BV605 (BD 563015) and anti-CD3 PECy7 (Biolegend, 100219). FACS was performed on a BD FACS Aria using FACSDiva Software. cDNA libraries were prepared for sequencing using standard protocol for the 10x Single Cell instrument.