Fitc anti mouse cd8a antibody

The mice were divided into five groups, the PBS group (NS), the OVA (20 µg per mouse) mixed with free CpG (20 µg OVA plus 8 µg CpG per mouse, OVA/CpG) group, the OVA mixed with alum adjuvant (20 µg OVA plus 100 µg Alum adjuvant per mouse, OVA/Alum) group, the OVA (0.2 mg/mL) admixed with the Ncom Gel (20 µg OVA plus 100 µL Ncom Gel per mouse, OVA/Ncom Gel) group and the Ncom Gel only (100 µL Ncom Gel per mouse) group. Female C57BL/6 mice were immunized on the 1^st, 15^th and 22^nd days. Next, spleen single-cell suspensions were obtained from immunized mice seven days after the last immunization, seeded in 24-well plates, restimulated with OVA (50 µg/mL) and incubated for 72 h. After incubation, spleen cells were centrifuged and washed twice, then the cultures were collected for IFN-γ detection. The obtained cells were stained with FITC anti-mouse CD8a antibody and FITC anti-mouse CD4 antibody (Biolegend, San Diego, CA, USA), respectively. Then, the labeled cells were fixed with paraformaldehyde for 30 min and washed twice. Then, the fixed cells were permeabilized with Triton X-100 (Sigma-Aldrich, Saint Louis MO, USA) and stained with PE anti-mouse IFN-γ antibody (Biolegend, San Diego, CA, USA). Finally, the cells were resuspended and analyzed by flow cytometry (ACEA NovoCyte, San Diego, California, USA).

The antigen-specific CD4⁺ and CD8⁺ T-lymphocyte responses to each immunized group were measured through an intracellular cytokine staining (ICS) assay. Briefly, 1 × 10⁶ splenocytes were seeded in a 6-well plate and treated with M. hyopneumoniae protein at a concentration of 10 μg/mL. Cells were labeled with APC anti-mouse CD3 antibody (Biolegend, San Diego, CA, USA), PE anti-mouse CD4 antibody (Biolegend), and FITC anti-mouse CD8a antibody (Biolegend) (PBS:CD3:CD4:CD8a = 377:10:5:8) at 4 °C for 25 min, away from light. After being washed twice with cold PBS buffer, cells were resuspended in PBS and analyzed using Accuri C6 Plus Flow Cytometry (BD Biosciences, Franklin Lakes, NJ, USA).