Apc fire750 labeled anti mouse cd8

OT-1 CD8⁺ T cells expressing a transgene encoding a TCR specifically recognizing SIINFEKL peptide bound to mouse H-2K^b were harvested from spleens of OT-1 C57BL/6 mice.^{31 (link)} Activated OT-1 T cells were generated by incubation of 5 × 10⁶ cells/ml OT-1 SIINFEKL-pulsed mouse splenocytes in vitro for 5–7 days in the presence of Mouse recombinant IL2.^{41 (link)42 (link)} Briefly, an OT-1 mouse spleen was harvested and homogenized using aseptic techniques. The released cells were pelleted and re-suspend in 3 ml ACK buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA) for 2 minutes to lyse red blood cells at room temperature. The splenocytes were then pelleted, washed, and re-suspended at 5 × 10⁶ cells/ml in complete growth medium (RPMI1640 Sigma-Aldrich) with 10% FBS (Corning), 1× penicillin-streptomycin (ThermoFisher Scientific), 1× sodium pyruvate (ThermoFisher Scientific), and 1× 2-Mercaptoethanol (ThermoFisher Scientific) containing 0.75 μg/ml SIINFEKL peptide (GenScript), and incubated at 37°C in a 95% air/5% CO2 humidified environment. Mouse recombinant IL2 (ThermoFisher Scientific) was added on days 3 and 5 at 30 U/ml with fresh complete growth medium. On day 7, the cells were harvested for assays. The specificity was determined by flow cytometry analysis using APC/Fire750-labeled anti-mouse CD8 and APC-labeled anti-mouse TCRVβ5.1/5.2 antibodies (BioLegend).