Anti cd3 anti cd28 activation beads

8–12 weeks old BALB/c or Nur77-GFP reporter mice were sacrificed, and spleen and inguinal and axillary lymph nodes were collected. Spleen and lymph nodes were crushed and filtered through a 70 μm nylon cell strainer (Corning). Red blood cells were lysed by ACK lysing buffer (A1049201, Thermo Fisher) CD8⁺ T cells were isolated using a negative CD8α⁺ T cell isolation kit (Cat#130–104-075, Miltenyi Biotec) and then cultured at 1 ×10⁶/mL in the presence of plate-coated anti-CD3/anti-CD28 crosslinking antibodies (anti-CD3 2 μg/mL; anti-CD28 2 μg/mL; Supplementary Table S1) in the presence or absence of 10 μM isoproterenol during activation (Sigma) as described previously (18 (link)). For immunoblotting (described below), CD8⁺ T cells were cultured at 2 ×10⁶/mL in the presence of 10 uL anti-CD3/anti-CD28 activation beads (Cat#11452D, ThermoFisher) and harvested at the indicated time points.