Cd4 fitc rpa t4

Flow cytometry analysis was performed to identify PBMC subpopulations and surface expression of CCR6. One million cells from each of the 40 subjects were incubated with human Fc Receptor binding inhibitor from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min. Cell were further stained for 30 min at room temperature with the following anti-human antibodies: CD8a PerCP (HIT8a), CD14 Pacific Blue (HCD14) and CD19 APC (HIB19) from BioLegend (San Diego, CA, USA), CD4 FITC (RPA-T4) from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) and CCR6 PE (11A9) or isotype control IgG1κ PE (MOPC-21) and CD16 APC-H7 (3G8) from BD Bioscience (Franklin Lakes, NJ, USA). After incubation with fix/lysis buffer from eBioscience (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at room temperature, cells were washed twice in FACS buffer (PBS with 0.1% BSA) and resuspended in PBS. Compensation controls were prepared using Anti-mouse Igκ/Negative Control Compensation Particle Set from BD Bioscience (Franklin Lakes, NJ, USA) according to the manufacturer’s recommendations. Flow cytometry was performed on a BD FACS Canto II flow cytometer with FACS Diva software from BD Bioscience (Franklin Lakes, NJ, USA) (10,000 events per sample) and samples were analysed using FlowJo v10 software from Flow Jo, LLC (Ashland, OR, USA).