Pe cy7 anti cd8α

BALF cells, mLN, or lung single cell suspensions were resuspended in cell staining buffer (no. 420201, BioLegend, San Diego, CA, USA) and blocked with TruStain FcX (anti-mouse CD16/32) antibody (no. 101320, BioLegend, San Diego, CA, USA) for 10–15 min on ice. The cells were then incubated 30 min with the following specific fluorochrome-conjugated antibodies or isotype controls (BioLegend, San Diego, CA, USA ) in the dark at 4 °C: Alexa Fluor-488 anti-CD45, PE anti-CD3, APC anti-CD4, FITC anti-CD4, PE-Cy7 anti-CD8α, APC anti-glycosylated CD43 (1B11), FITC anti-CD43, FITC anti-CD44, APC anti-CD11a, PE/Cy5 anti-CD69, Pacific Blue anti-CD54, APC anti-CD11b, PE anti-Ly6G, or FITC isotype control antibodies. PE-conjugated tetramer specific for H-2Db IAV NP_366–374 (ASNENMETM) was from MBL International Corporation (Woburn, MA, USA). After washing, stained cells were acquired in an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA USA). Cell death was determined by flow cytometry using APC annexin V apoptosis detection kit with propidium iodide (no. 640932, BioLegend, San Diego, CA, USA). Flow cytometry data were analyzed by using FlowJo software v10.6.2.

For analysis of cytotoxic T cell infiltration in mouse tumors, mouse tumors were digested at 37 °C at 100 rpm for 1 h in digestion buffer (RPMI 1640 medium containing 0.4 mg/mL Collagenase IV (Sigma-Aldrich) and 50 U/mL DNase I (Sigma-Aldrich). The digested cell suspension was filtered by a 70-μm cell strainer to obtain single cell suspension and washed with staining buffer (PBS containing 2% FBS) twice and PBS once. Lymphocytes were obtained from peripheral blood lymphocyte isolates (TBD, LTS1077). Then cells were stained with Zombie Aqua™ Fixable Viability Kit (bv510) (Biolegend, 423101) in PBS for 15 min to stain the non-viable cells. After that, cells were washed with staining buffer twice and stained with APC anti-CD45 (Biolegend, 103111), FITC anti-CD3ε (Biolegend, 100203), and PE/cy7 anti-CD8α (Biolegend, 100721) antibodies at 4 °C for 30 min. Flow cytometry analysis was performed after cells were washed twice with staining buffer.