Immunocult xf t cell expansion media

Human peripheral blood CD8⁺ T cells (Stem Cell Technologies, 200–0164) were cultured in ImmunoCult XF T Cell Expansion media (Stem Cell Technologies, 10981) at 37 °C with 5% CO₂. Antibodies were immobilized on Dynabeads M-280 Tosylactivated (Invitrogen, 14203) as described previously (Desai et al., 2022). Briefly, a total of 10 μg of antibody consisting of 2 μg of anti-human CD3 antibody (Bio X Cell, BE0001–2) and 8 μg of experimental antibody in 1 mL of 1X PBS was incubated at room temperature for 2 days with rotation. The beads were subsequently blocked with 10 mM glycine buffer for 1 h with rotation, followed by washing twice with PBSB. Next, the CD8⁺ T cells were plated at a density of 50,000 cells/well in a 96-well plate to a volume of 150 μL. Then, 50 μL of bead solution was added at a ratio of 4:1 or 8:1 of conjugated beads:T cells. After incubating at 37 °C with 5% CO₂ for 96 h, the cells were stained with BV510 anti-human CD8 (BioLegend, 344732) and APC anti-human CD25 (BioLegend, 302610) antibodies at 1:200 dilution each on ice for 5 min. Cells were then centrifuged at 300 xg for 4 min, washed with 1X PBS, and analyzed via flow cytometry.