Cd44 bv421

T cells were washed with PBS containing 2% FBS prior to the addition of antibodies. Cells were stained with fluorophore-conjugated antibodies specific for mouse CD3-AF700 (BD Biosciences, San Jose, CA, USA), CD8-APC (eBioscience, Waltham, MA), CD27-PE (BD Biosciences, San Jose, CA, USA), and CD44-BV421 (BD Biosciences, San Jose, CA, USA) in PBS with 2% FBS for 15 min at 4 °C. Cells were then fixed in 4% paraformaldehyde for 10 min at 4 °C and washed twice in PBS with 2% FBS. Flow cytometry was performed on a Canto II flow cytometry system (BD Biosciences, San Jose, CA) 32 (link).

An amount of 2 μg of anti-mouse CD45-BV786 antibody (BD Biosciences, Franklin Lakes, NJ, USA) was diluted in sterile PBS and administered intravenously in the tail vein 10 min before anesthesia. CD 45+ is a circulating lymphocyte, and CD45− is a tissue-resident cell. In total, 40 U/mL DNase I and 1 mg/mL Collagenase IV enzyme were measured to a volume of 2 mL and incubated with lung tissue at 37 °C for 30 min. The tissue was then homogenized using a dissociator, filtered through a 70 μM cell strainer, and subjected to RBC lysis to obtain single lung cells. After preparing the lung cells to a concentration of 5 × 10⁶ cell/mL, they were washed with PBS and live-cell staining was performed using fixable viability stain 700 (FVS700) (BD Biosciences, Franklin Lakes, NJ, USA). After treating lung cells with Fc Block^TM reagent, the surface markers CD44-BV421, CD69-BV605, CD103-PE, CD62L-APC, CD4-FITC, CD3-PE-Cy7, and CD8-BB700-per cycle 5.5 ((BD Biosciences, Franklin Lakes, NJ, USA)) were used for staining. The flow cytometry gating strategy was performed according to Supplementary Materials Figure S1, based on a previous study [45 (link)]. FACS Aria Fusion from BD Biosciences was used, and all results were derived using FlowJo software v10.