V500 conjugated anti cd4

Splenocytes from each immunized mouse were seeded into 96-well plates (1 × 10⁶ cells/well) in complete RPMI-1640 medium and stimulated with RBD protein (Sino Biological, Beijing, China; 5 μg/ml) in a total volume of 200 μl for 20 h. Stimulation with PMA (50 ng/ml) and ionomycin (1 μg/ml) was used as a positive control. To prevent cytokine release, brefeldin A (10 μg/ml) was added, and the cells were cultured for an additional 4 h. Cell surface markers were stained with FITC-conjugated anti-CD3, V500-conjugated anti-CD4 and PE-conjugated anti-CD8 (BD, New Jersey, USA) antibodies. After being fixed with 1% paraformaldehyde (20 min, RT) and permeabilized with 0.1% Triton X-100 (30 min, RT), the cells were stained intracellularly with APC-conjugated anti-IFN-γ (BD, New Jersey, USA) antibody. The stained cells were analyzed using a FACS LSRFortessa instrument (BD, New Jersey, USA) and the FlowJo program.