Anti mouse cd8a efluor 450

Spleens were excised from 6 – 12-week-old C57BL/6 mice and put in 5 ml of FACS buffer. Each spleen was then transferred to a 100 μm cell strainer atop a 35 mm plate containing 2 ml of FACS buffer. The plunger of a small syringe was used to crush the spleen in the strainer. The cell suspension from the plate beneath was collected and briefly centrifuged. Red blood cells were removed from the cell pellets by resuspension in 1 ml of ACK lysis buffer (#10-548E; Lonza) for 1 minute. After that, AKC buffer was diluted by adding 9 ml FACS buffer. The cells were then harvested, washed one more time, and resuspended at a concentration of 1 × 10⁶ cells/ml in FACS buffer. Mouse spleen CD8⁺ T cells were enriched by magnetic cell sorting using negative selection kits (#130-104-075; Miltenyi Biotec). The purity of the CD8⁺ cell populations was determined using flow cytometry by staining with anti-mouse CD8a eFluor 450 (#48-0081-82; dilution 1:200; eBioscience). Mouse CD8⁺ T cells were cultured in TexMACS media (#130-097-196; Miltenyi Biotec) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/mL streptomycin. 400 ng/mL anti-mouse CD3 (#50-139-2707, Fisher Scientific), 400 ng/mL anti-mouse CD28 (#50-562-020, Fisher Scientific), and 100 IU/mL recombinant mouse IL-2 (#PMC0025; Gibco) was freshly added to the medium each time used.