Easysep mouse naive cd8 t cell isolation kit

Mouse naive CD8⁺ T cells were negatively selected by EasySep Mouse Naive CD8⁺ T Cell Isolation Kit (STEMCELL). Isolated naive CD8⁺ T cells were cultured in RPMI-1640 medium with 10% FBS, 1% penicillin, and 100 μg/mL streptomycin in flat-bottom plates precoated with anti-CD3 (2 μg/mL; clone 145-2C11; eBioscience) and anti-CD28 (2 μg/mL; clone 37.51; eBioscience) for 2 days, unless otherwise stated. For small molecular inhibitor treatment, Stattic (Selleck, S7024), SH-4-54 (Selleck, S7337), STAT5-IN-1 (Selleck, S6784), or 17-AAG (Selleck, S1141) was added to culture medium and cells were cultured for indicated times.

CD8⁺ T cells were isolated from smashed spleens and inguinal lymph nodes of P14 Nur77-GFP mice using the EasySep™ Mouse naive CD8⁺ T-cell Isolation Kit (StemCell, Grenoble, France) following the manufacturer’s instructions. Spleens and lymph nodes were smashed using a 70 µm cell strainer. Isotopically labeled mouse DCs were incubated with no compound or 1 μg/ml purified gp33 carrying no (gp33), one (gp33-SOG), or two (gp33-SOG₂) SOGs for 1 h. A 10-fold excess of acutely isolated CD8⁺ T cells was added by centrifugation and cells were co-incubated for 3 h in the presence of interleukin-2. Cells were carefully washed with ice-cold PBS, chilled photo-oxidation buffer was added and interacting cells were illuminated at a working distance of 20 cm with 590-nm Precision LED spotlights for 15 min. Cells were pelleted by centrifugation and resuspended in chilled labeling buffer for 50 min at 4 °C in the dark. Cells were extensively washed with PBS and assessed for T-cell activation and cell-type-specific surface biotinylation using flow cytometry.