Ifn γ alexa fluor 700

The following fluorochrome-conjugated Abs were used for flow cytometry analysis: HIV-p24 FITC (KC57) (Beckman Coulter, Fullerton, CA, USA), HIV-p24 PE (KC57) (Beckman Coulter, Fullerton, CA, USA), CD3 Pacific blue (UCHT1), CD4 PerCP/Cy5.5 (RPA-T4) (Biolegend, San Diego, CA, USA), CD4 Alexa Fluor 700 (RPA-T4), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), RORC2 Alexa Fluor 647 (Q31-378), Ki-67 BUV395 (B56), IL-17A PE (eBio64DEC17) and IFN-γ Alexa Fluor 700 (B27). Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid, Life Technologies, Burlington, Ontario, CA) was used to exclude dead cells. Intracellular staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences) and intranuclear staining was performed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set. Cells were analysed with the BD-LSRII cytometer, BD LSRFortessa and BD-Diva (BD Biosciences) and FlowJo version 10 (Tree Star, Inc., Ashland Oregon, USA). The positivity gates were placed using fluorescence minus one (FMO) strategy (8, 20) . For FACS, memory CD4 T cells from PBMCs of ART+ PLWH were isolated by negative selection using magnetic beads. CCR6 + RORC2+, CCR6+RORC2-and CCR6 -RORC-T cells were sorted by FACS (BDAria II; BD Biosciences) using the antibodies CD3 Pacific blue (UCHT1), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), and RORC2 Alexa Fluor 647 (Q31-378).