Membrane washing buffer

The splenocytes from the immunized mice were grown on 12-well plates. Phorbol 12-myristate 13-acetate (PMA; Sigma, San Francisco, CA, USA), ionomycin (Solarbio, Beijing, China), and brefeldin A (Solarbio, Beijing, China) were mixed to stimulate the cell at 37 °C with 5% CO₂ for 6 h. After centrifugation, the cells were washed with PBS, and stained with CD4-488 (1:200; BioLegend, San Diego, CA, USA) and CD8-APC (1:300; BioLegend) for 1 h in the dark. Then, 100 µL of fixative (BioLegend) was added to fix the cells for 20 min at 4 °C in the dark. A membrane washing buffer (BioLegend) was added to wash and resuspend the cells. Cells were incubated overnight at 4 °C with IFN-γ-PE (1:200; BioLegend) diluted in a transmembrance washing solution. The analysis of the proportion of IFN-γ among gated CD4⁺ and CD8⁺ T cells was performed by flow cytometry (BD, Franklin Lakes, NJ, USA).

To measure IFN-γ levels by stimulating splenocytes in vitro, splenocytes were cultured in 12-well plates containing proteins of rPoAMA-1. After 18 h, the cells were simultaneously mixed with various concentrations of phorbol 12-myristate 13-acetate (50 ng/mL PMA; Sigma), ionomycin (1 µg/mL; Solarbio), and brefeldin A (5 µg/mL; Solarbio) for 6 h at 37 °C with 5% CO₂. The cells were washed twice with PBS. Subsequently, 50 µL PBS containing CD4–488 (1:200; BioLegend, California, USA) and CD8-APC (1:300; BioLegend, California, USA) were added to samples at 4 °C for 1 h, and then centrifuged (1500 × g, 10 min) and washed twice with PBS. Then, 100 µL of fixative (BioLegend, California, USA) was added, and the samples were incubated in a dark environment at 4 °C for 20 min. The samples were then washed with membrane washing buffer (BioLegend, California, USA), and 50 μL of IFN-r-PE (1:200; BioLegend, California, USA) diluted in transmembrane washing solution was added in a dark environment at 4 °C overnight. Finally, the samples were analyzed in BD Accuri C6 PLUS flow cytometry (BD, New Jersey, USA).