Anti cd4 conjugated to bv786

ICS was performed on splenocytes isolated from immunized BALB/c mice. For the assay, 2 × 10⁶ cells were plated into the wells of 24-well culture plates (TPP, Trasadingen, Switzerland) and stimulated with the peptide mixture indicated above. Each peptide was added at a concentration of 20 μg/mL per well, and cells were incubated for 4 h at 37 °C in the presence of 5% CO₂ and for an additional 16 h with Brefeldin A (5 μg/mL, GolgiPlug BD Biosciences). The next day, the cells were stained with anti-CD3 conjugated to Alexa Fluor 700 (BD), anti-CD4 conjugated to BV786 (BD), and anti-CD8 conjugated to FITC (BD); fixed with 1% paraformaldehyde in PBS; and permeabilized with 0.5% Tween 20 in PBS according to the manufacture’s instructions. Then, the cells were stained to detect intracellular cytokines anti-IFN-γ APC (BD, USA). The samples were analyzed by using a ZE5 flow cytometer (Bio-Rad) and the Everest software.

Splenocytes were plated at 2 × 10⁶ cells/well into the 24-well culture plates (TPP, Trasadingen, Switzerland) and stimulated with the pool of peptides mentioned above. Cells were incubated for 4 h at 37 °C in the presence of 5% CO₂ and for an additional 16 h with Brefeldin A (5 μg/mL, BD Biosciences, CA, USA). The next day, the cells were stained with anti-CD3 conjugated to Alexa Fluor 700 (BD), anti-CD4 conjugated to BV786 (BD), and anti-CD8 conjugated to FITC (BD); fixed with 1% paraformaldehyde in PBS; and permeabilized with 0.5% Tween-20 in PBS according to the manufacturer’s instructions. Then, the cells were stained with anti-IFN-γ APC (BD Biosciences, CA, USA) and analyzed using a ZE5 flow cytometer (Bio-Rad, Hercules, CA, USA) and the Everest software.