α cd8a efluor450 okt8

BAL and NT samples were stained in 96-well plates using the Forkhead Box P3 (FoxP3)/transcription factor staining buffer set (eBioscience, Thermo Fisher Scientific). Cells were stained with α-CD4–allophycocyanin (02, Sino Biological, Beijing, China), α-CD8a–eFluor450 (OKT8, eBioscience), α-CD14–phycoerythrin (Tük4, Thermo Fisher Scientific), and Fixable Viability Stain 780 (BD, Franklin Lakes, NJ) in 100 μl for 30 min at 4°C. Samples were then washed twice with 150 μl of FACS buffer, followed by fixation with 100 μl of fixative from the FoxP3 staining kit for 20 min at room temperature. Next, samples were washed twice with 150 μl of 1× permeabilization buffer (FoxP3 staining kit). After the second wash, samples were stained with 100 μl of permeabilization buffer containing α-CD3e–fluorescein isothiocyanate (CD3-12, Bio-Rad) for 30 min at 4°C. Samples were then washed twice with 150 μl of 1× permeabilization buffer and once with 150 μl of FACS buffer. After the last wash, samples were resuspended in 180 μl of FACS buffer, after which 50 μl of precision count beads (BioLegend, San Diego, CA) were added to BAL and NT samples. Samples were measured in plates using the high-throughput system of a Symphony A3 system (BD). Data were analyzed using FlowJo Software v.10.6 (BD).