Cd127 af647

For TCR sequencing purposes, CD3⁺CD4⁺CD25^highCD127^low Tregs and CD3⁺CD4+CD25^low/intCD127^int/high non-Tregs were isolated from frozen PBMCs and SFMCs, using the FACS Aria III (BD, Franklin Lakes, NJ). Antibodies used for sorting were: anti-human CD3-BV510 (BioLegend, San Diego, CA), CD4-FITC (eBioscience, Frankfurt am Main, Germany), CD25-PE/Cy7 (BD), and CD127-AF647 (BioLegend). To check for FOXP3 expression of the sorted populations anti-human FOXP3-eF450 (eBioscience) was used.

Stimulation of cell culture was carried out with 1 μg/ml protein A (SpA, isolated from S. aureus SAC, GE Healthcare, Uppsala, Sweden), 1 µg/ml recombinant SpA (Sigma-Aldrich, Munich, Germany), 100 ng/ml synthetic lipopeptide P3C (Pam3CSK4, EMC, Tübingen, Germany) or anti-CD3/CD28 microbeads (Miltenyi Biotech, Bergisch-Gladbach, Germany).
Microbeads used for cell isolation via AutoMACS and recombinant cytokines (IL-4 and GM-CSF) were obtained from Miltenyi Biotech (Bergisch-Gladbach, Germany).
Antibodies used for flow cytometry, purity determination and cell sorting were purchased from BD Biosciences, Heidelberg, Germany, if not indicated otherwise: CD4 PErCP, CD4 PE, CD3 BV605 (BioLegend, U.S.), CD3 AF700 (BioLegend, U.S.), CD25 APC (BioLegend, U.S.), CD25 FITC, CD127 BV421, CD127 AF647, FoxP3 BV421 (BioLegend, U.S.), CCR4 PE-Cy7 (BioLegend, U.S.), ICOS BV605 (BioLegend, U.S.), CTLA4 BV421 (BioLegend, U.S.), PD-1 PE (BioLegend, U.S.) and CD14 V450. Viability staining was performed with the LIVE/DEAD Fixable dead Cell stain Kit (Thermo Fisher Scientific, U.K.).