Alexa fluor 700 conjugated cd8

For phenotypic characterization, 3 × 10⁵ cells were stained with fluorescein-isothiocyanate (FITC) anti-CD3, peridinin chlorophyll (PerCP)-conjugated anti-CD4, alexa fluor (AF) 700-conjugated CD8, brilliant violet (BV) 510-conjugated anti-CD45RA, allophycocyanin/Cyanin 7 (APC/Cy7)-conjugated anti-CD62L, anti-CD45RO-phycoerythrin (PE), and AF647-conjugated anti-CD197 (all BioLegend, London, Great Britian) monoclonal antibodies for 20 min at room temperature in the dark, washed with PBS (Lonza, Verviers, Belgium) with 0.1% human AB serum (C.C. pro, Oberdorla, Germany) and analyzed by multicolor flow cytometry (FACS Canto II, FACSDiva V8.1.2 software, BD Biosciences, Heidelberg, Germany). Gates were set based on the forward scatter versus side scatter properties of lymphocytes. At least 30,000 events were acquired in the CD3⁺ gate. For detailed gating strategy see Supplementary Figure S1B,C. For intracellular staining (ICS) peridinin chlorophyll (PerCP)-conjugated anti-CD3, alexa fluor (AF) 700-conjugated CD8, additional fluorescein isothiocyanate (FITC)-conjugated anti-IFN-γ and APC-conjugated TNF-α antibodies were used (both BioLegend). To determine Tregs and γδ T cells, alexa fluor (AF) 700-conjugated CD4, BV421-conjugated anti-CD25 (BioLegend), APC-conjugated anti-CD127, and anti-γδ TCR-PE/Cy7 antibodies were used (both BD Biosciences).