Cd103 biotin

For cell surface staining, empirically determined dilutions of primary mAbs were used to stain single cell suspensions on ice for 20 min in FACS buffer (PBS with 2% FBS, 0.1% NaN₃, and 1 µM EDTA). All mAbs were purchased from BioLegend unless otherwise indicated. The following mAb clones were used: B220-PE-Cy7, CD4-PE-Cy7, CD8α-PerCP-Cy5.5, CD11b-PE-Cy7, CD11c-FITC, CD25–Alexa Fluor 647, CD44-FITC, CD45.1-FITC, CD45.2-BV605, CD62L-PE-Cy7, CD103-biotin (followed by streptavidin-BV711; BD), Foxp3-PE (eBioscience), Helios-APC, I-A(b)-PE, Ki-67–Alexa Fluor 647, and TCR-β–Pacific blue. Dead cells were excluded using Fixable Viability Dye eFluor780 (eBioscience). Intracellular staining to detect Foxp3 expression was performed according to the manufacturer’s instructions, using the manufacturer’s protocol for Foxp3 staining in 96-well plates (eBioscience). Cells were analyzed using an LSR-II flow cytometer (BD) equipped with 405-, 488-, 552-, and 640-nm lasers. Flow cytometry data were processed using FlowJo version 9.7.5 software (Tree Star).