Percpvio700 labeled anti cd4

We performed FACS analysis to determine the proportion of cluster of differentiation (CD) 4⁺ and CD8⁺ cell subpopulations in the immunized mice. Briefly, 14 days post-immunization, a single-cell suspension of splenocytes was prepared (n = 5), and the cell number was adjusted to 1 × 10⁶ /mL. Splenocytes were cultured in 10% RPMI medium in 96-well plates (1 × 10⁵ cells/well). Splenocytes were stimulated with LI-specific recombinant proteins (500 ng/well) or RPMI medium alone for 24 h at 37°C in a 5% carbon dioxide atmosphere and stained for CD3, CD4, and CD8 markers using PE-labeled anti-CD3e, PerCPVio700-labeled anti-CD4, and fluorescein isothiocyanate-labeled anti-CD8a (Miltenyi Biotec, Germany). The CD3⁺ T-cell population was gated to analyze the CD3⁺CD4⁺ and CD3⁺CD8⁺ subpopulations.

Splenocytes harvested from the mice were analyzed for viability by diluting the samples in 0.4% trypan blue solution at a 1:1 ratio. Then, 3 × 10⁶ cells per tube were stained for cell surface CD3, CD4, and CD8 markers using the following monoclonal antibodies: PE-labeled anti-CD3e, PerCPVio700-labeled anti-CD4, and FITC-labeled anti-CD8a (Miltenyi Biotec, Germany). Cells were gated on the basis of their forward and side scatter profiles.