Mca1477t

Seven weeks after immunization, blood from each group was collected into anticoagulant tubes containing heparin sodium, 20 times the volume of whole-blood red blood cell lysis buffer (BioLegend, 420,301, USA) was added, and the samples were lysed for 10 min at room temperature. After lysis, the cells were centrifuged at 400 g for 5 min at 4 °C. Leukocytes were resuspended in PBS and counted. Then, 0.2 μg of mouse anti-feline CD4-PE and CD8-FITC antibodies (SouthernBiotech, 8130–09, 8120-02, USA) were added to each sample of 10⁶ cells, and the resulting mixture was incubated at 4 °C in the dark for 30 min and washed twice with PBS. Surface antigen fixation and membrane permeation of CD4+ and CD8+ cells were performed by adding fixation buffer (BioLegend, 420,801, USA) and intracellular staining permeabilization wash buffer (BioLegend, 421,002, USA). The anti-CD3–647 (Bio-Rad, MCA1477T, USA) antibody was added, and the samples were incubated for 30 min at 4 °C in the dark and washed twice with wash buffer. The cells were resuspended in 500 μl of buffer and filtered through a 70-μm cell mesh sieve. Flow cytometry (Beckman Coulter, Cytoflex, USA) was performed, and the results were analysed with FlowJo software.