Pe anti mouse cd4 antibody

Splenic lymphocyte CD4⁺/CD8⁺ subclass analysis was performed using flow cytometry. An amount of 97 µL of cell staining buffer (BioLegend, San Diego, CA, USA, Cat. No#420201) and 0.5 µL of TruStain FcXTM PLUS (anti-mouse CD16/32) (BioLegend, Cat. No#156604) antibody were transferred to 1.5 mL Eppendorf (EP) tubes containing 1.5 × 10⁶ cells, mixed softly, and then used to stain the cells for 10 min at RT in the dark. Thereafter, 0.5 µL of FITC anti-mouse CD3ε antibody (BioLegend, San Diego, CA, USA, Cat. No#100204), 1.25 µL of PerCP/Cyanine5.5 anti-mouse CD8α antibody (BioLegend, San Diego, CA, USA, Cat. No#100734), and 1.25 µL of PE anti-mouse CD4 antibody (BioLegend, San Diego, CA, USA, Cat. No#100512) were added to the EP tubes, mixed softly, and then used to stain the cells for 30 min at 4 °C in the dark. Then, the EP tubes were centrifuged at 200× g for 5 min, and the supernatant was discarded. Next, 1 mL of PBS was added to make a cell suspension, centrifuged at 4 °C, 200× g for 5 min, and the supernatant was then removed. Cells were centrifuged twice, and 250 µL of cell staining buffer was added for cell suspension. Finally, the cells were analyzed by FACSVerse flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA), and all dates were analyzed using FlowJoV_10 software.

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).