Pe conjugated anti mouse cd95

The mice were humanely sacrificed, and the spleens were removed. The spleens were triturated, and the red blood cells were removed using RBC Lysis Buffer (Solarbio) according to the kit instructions. After centrifuging at 500 g for 10 min at 4°C, the supernatants were discarded, and cells were resuspended and washed twice with Staining buffer (eBioscience) to obtain single-cell suspensions. Then, cells were stained with different combinations of flow cytometry antibodies, including APC-conjugated anti-mouse CD3 (eBioscience, San Diego, United States), FITC-conjugated anti-mouse CD4 (BioLegend, San Diego, United States), PE-conjugated anti-mouse CD8 (eBioscience) APC-conjugated anti-mouse B220 (BioLegend), Pacific Blue-conjugated anti-mouse GL-7 (BioLegend), PE-conjugated anti-mouse CD95 (BioLegend), PE-conjugated anti-mouse PD-1 (BioLegend), and Brilliant Violet 421-conjugated anti-mouse CXCR5 (BioLegend). After staining, cells were washed with Staining buffer and dispersed in 500 mL of Staining buffer. Analysis was performed using a Mona CytoFLEX flow cytometer (BeckmanCoulter LifeSciences, Brea, United States).

For cell surface marker staining, draining lymph nodes (dLNs) of each mouse were individually collected and triturated into a single cell suspension. Then the cells were stained with different combinations of flow cytometry antibodies for 30 min at 4°C, which included APC-conjugated anti-mouse CD3 (eBioscience, USA), eFluor450-conjugated anti-mouse CD4 (eBioscience, USA), PE-conjugated anti-mouse CD8a (eBioscience, USA), APC-conjugated anti-mouse B220 (Biolegend, USA), Pacific Blue-conjugated anti-mouse GL-7 (Biolegend, USA), PE-conjugated anti-mouse CD95 (Biolegend, USA), FITC-conjugated anti-mouse CD4 (Biolegend, USA), PE-conjugated anti-mouse PD-1 (Biolegend, USA), and Brilliant Violet 421-conjugated anti-mouse CXCR5 (Biolegend, USA). After washing with staining buffer (eBioscience, USA), the cells were dispersed in 500 μL of staining buffer (eBioscience, USA) and analyzed by flow cytometry (Beckman Coulter, Cytoflex LX).

Vaccine candidates NP‐OPS_KpO2, C‐OPS_KpO2, and OPS_KpO2 were each prepared at the same dose. All candidates and PBS control were subcutaneously injected into the tail base of BALB/c mice. Some of the treated mice were sacrificed on the 3rd day postinjection and analyzed the proportions of CD4⁺ and CD8⁺ T cells in their dLNs via FCM. Moreover, on the 7th day postinjection, the proportions of Tfh cells, and B cells in GCs were analyzed.
First, isolated popliteal dLNs to precooled PBS and triturated them into single‐cell suspensions. Then, cells were stained with different combinations of FCM antibodies, including APC‐conjugated anti‐mouse CD3 (eBioscience, San Diego, USA), FITC conjugated anti‐mouse CD4 (BioLegend, San Diego, USA), PE‐conjugated anti‐mouse CD8a (eBioscience), PE conjugated anti‐mouse PD‐1 (BioLegend), Brilliant Violet 421 conjugated anti‐mouse CXCR5 (BioLegend), APC‐conjugated anti‐mouse B220 (BioLegend), Pacific Blue conjugated anti‐mouse GL‐7 (BioLegend), and PE conjugated anti‐mouse CD95 (BioLegend) for 30 min at 4 °C. After washing them with staining buffer (eBioscience), the cells were dispersed in 500 µL PBS and analyzed them on a CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences, Brea, USA).