Anti cd4 buv563

PBMCs from four BCG-vaccinated healthy controls and P were stained with the CellTrace Far-Red Cell Proliferation Kit (Thermo Fisher Scientific) at a dilution of 1:25,000. Cells were washed and resuspended at a density of 2 x 10⁶ cells/mL in RPMI 10% human AB serum (Gemini). Cells were plated in 96-well U-bottomed plates and stimulated with BCG-lysate at a final concentration of 5 μg/mL or with tuberculin purified protein derivative (PPD) at a final concentration of 5 μg/mL (STATEN SERUM INSTITUT). Cells were cultured for 6 days and then harvested and stained with the Zombie NIR Viability kit (BioLegend) for 15 minutes. Cells were then surface-stained with FcBlock, anti-CD3-V450 (BD Biosciences), anti-CD4-BUV563 (BD Biosciences), anti-CD8-BUV737 (BD Biosciences), anti-Vδ2TCR-APC/Fire750 (BioLegend), anti-CD56-BV605 (BioLegend) and anti-CD20-BV785 (BioLegend) antibodies for 30 minutes. Cells were fixed with the FOXP3/Transcription Factor Buffer kit (Thermo Fisher Scientific) and stained by overnight incubation with anti-T-bet-PE/Cy7 (BioLegend), anti-IFN-γ-BV711 (BioLegend), anti-TNF-α-BV510 (BioLegend) and anti-human/mouse-Ki-67 (BioLegend) antibodies in Perm/Wash buffer. Data were acquired in a CyTek Aurora spectral flow cytometer.