Anti mouse cd16 cd32 mouse fc block mab clone 2.4g2

Anti-CD8α mAb (clone 53–6.7, 10 µg) or anti-CD4 mAb (clone GK1.5) was administered intravenously 48 hours prior to tumor implantation and subsequently every 2 days using a low dose of 10 µg. Depletion of CD8⁺ or CD4⁺ T cells was confirmed 48 hours post first administration and on day 12. Blood (150 µL) was collected from the tail vein in 500 µL 1× PBS containing heparin and kept on ice. Cells were centrifuged at 1200 rpm for 5 min. RBCs were lysed using 500 µL ACK buffer (Sigma-Aldrich) on ice for 2–3 min. Cells were collected by centrifugation for 5 min at 1200 rpm. The supernatant was aspirated and cells were resuspended with 1× PBS containing 0.1% BSA and centrifuged again. Pelleted cells were stained with anti-mouse CD16/CD32 (mouse BD Fc block) mAb clone 2.4G2 (BD Bioscience) for 10 min in the dark, centrifuged as described earlier, and stained with PE anti-mouse CD8α mAb clone 53–6.7 (BD Bioscience) or FITC anti-mouse CD4 mAb clone (RM4-5). Samples were read by a FACSCaliber and analysis was done using GraphPad Prism V.8.4.2 (464).