Brilliant violet 510 conjugated cd8a

In order to analyze multifunctional antigen-specific T cells, freshly isolated splenocytes (1.5 × 10⁶) were seeded in 96-well plates and stimulated with 5 μg/mL Ag85B or 1 μ/mL ESAT-6 antigens. As a positive control, cells were stimulated with 100 ng/mL PMA and 1 μg/mL Ionomycin. After 4 h incubation, cells were blocked and fixed, as described in Flow cytometry of mouse cells paragraph. Subsequently, cells were stained with FITC-conjugated CD3, PerCP/Cy5.5-conjugated CD4, Brilliant Violet 510™-conjugated CD8a, APC-conjugated TNF-α, PE/Cy7-conjugated IL-17A, PE/Cy5-conjugated IL-2 and Brilliant Violet 421™-conjugated IFN-γ antibodies (all from BioLegends), all diluted 1:100, and analyzed by flow cytometry.

Freshly isolated splenocytes (1 × 10^E6) were seeded in 96-well plates and stimulated with 5 μg/mL Ag85B antigen or 1 μ/mL ESAT-6 antigen. As positive control, the cells were stimulated with 1 μg/mL α-CD3 (BioLegends). Antigen-specific T cell proliferation was analyzed after 6 days of incubation with antigens. The cells were blocked, as described above, and subsequently stained with PerCP/Cy5.5-conjugated CD4, Brilliant Violet 510™-conjugated CD8a, FITC-conjugated CD44, PE-conjugated CD62L and Brilliant Violet 421™- conjugated CD90.2 antibodies (all from BioLegends), all diluted 1:100. After staining, the cells were fixed using eBioscience™ Foxp3/Transcription Factor Staining Buffer Set and permeabilized using eBioscience™ Permeabilization Buffer, according to the manufacturer's instructions. Subsequently, cells were intracellularly stained with 1:50 diluted APC-conjugated Ki-67 antibody (BioLegends) and analyzed by flow cytometry.