Pma ionomycin

Splenocytes were suspended in 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin–streptomycin (Thermo Fisher) at a final concentration of 1 × 10⁷ cells/mL. Then, 1 × 10⁶ cells were added to each well of a 96-well plate (Corning Inc., Corning, NY, USA). With 10 μL of PMA + ionomycin (stock solution concentrations: 500 ng/mL + 10 μg/mL; DAKEWE, Beijing, China) as positive control, gE was added to each well at a final concentration of 10 μg/mL. After incubation at 37 °C for 24 h in a 5% CO₂ atmosphere, the supernatants of the cells were collected to test the contents of IL-2 and IFN-γ. Briefly, anti-IL-2 (3 μg/mL) and anti-IFN-γ (4 μg/mL) capture antibodies (Invitrogen, Carlsbad, CA, USA) dissolved in PBS were added to coat 96-well plates and incubated overnight at 4 °C. After blocking with 5% (w/v) skim milk at 37 °C for 1 h, 50 μL of cell supernatant was added to each well and incubated for another 3 h at room temperature. PBS-dissolved standard mouse IL-2 and IFN-γ proteins (PeproTech, Cranbury, NJ, USA) were used to generate standard curves. Biotin-conjugated detection antibodies specific for IL-2 or IFN-γ (2 μg/mL, Invitrogen, USA) and HRP-conjugated streptavidin (1 μg/mL, BioLegend, San Diego, CA, USA) were then added and incubated for 1.5 h. The results were detected as mentioned above in the ELISA analysis.

The levels of the cytokines IL-2 and IFN-γ secreted by mouse spleen cells were detected by ELISA with the double-antibody sandwich method. The capture antibody was diluted with PBS to the appropriate concentration (3 μg/mL for IL-2 and 4 μg/mL for IFN-γ). Prepared spleen cells were added to 96-well plates (1 × 10⁶ cells per well), and gE protein was added to the wells other than the negative and positive control wells as a stimulus. PMA + ionomycin (10 μg/mL, DAKEWE, Beijing, China) was added to the 96-well plates (Corning Inc., Corning, NY, USA) as a positive control. The cells were incubated overnight at 37 °C and 5% CO₂, and the cell supernatants were collected to detect the levels of IL-2 and IFN-γ [22 (link)].