Apc h7 conjugated anti cd8

Samples of 100 μL per tube blood were transferred for staining with monoclonal antibodies and red blood cell (RBC) lysis. RBCs were lysed with buffer containing 0.8% NH₄Cl and 0.1% KHCO₃. Cells were then washed with PBS (phosphate-buffered saline) and stained with FITC-conjugated anti-CD3 or anti-CD95, RPE-Cy5-conjugated anti-CD4 (BD Pharmingen, San Diego, CA, USA), APC-H7-conjugated anti-CD8, PE-conjugated anti-CD28, anti-CD25, anti-CD69, or anti-HLA-DR (BD Pharmingen, USA) for 30 min at 4 °C in the dark. Then, cells were washed with PBS and suspended in 200 mL of suitable buffer for flow cytometric analysis using a FACSVerse instrument (Becton Dickinson, Franklin Lakes, NJ, USA).

All antibodies were pre-titrated to determine appropriate working concentrations. Immunostaining with periridin-chlorophyll protein (PerCp)-Cy5.5-conjugated anti-CD3 (BD Biosciences, clone UCHT1), allophycocynanin (APC)-H7-conjugated anti-CD8 (BD Biosciences, clone SK1) and APC-conjugated anti-CD161 (BD Biosciences, clone DX12) in the phenotypic characterization of MAIT cell was performed according to the protocol set by the commercial manufacturer (BD Biosciences). Surface staining for specific receptors was performed using monoclonal antibodies (mAbs) directed against: FITC-conjugated PD-1 (clone MIH4), phycoerythrin (PE)-conjugated CCR6 (clone 11A9), PerCp-Cy5.5-conjugated CCR5 (clone 3A9) and PE-conjugated TCR Vα7.2 (clone 3C10) (all mAbs procured from BD Biosciences). Immunostained samples were washed twice prior to acquisition on a FACS Canto II Immunocytometry system (BD Biosciences) and analyzed using FACS Diva software. Data were analyzed using FlowJo (version 9.3.1 and version 10). Doublets were excluded based on FSC-H and FSC-A, lymphocytes were identified based on FSC and SSC, and dead cells were excluded based on BD Horizon Fixable Viability Stain 510 (BD Biosciences). CD3⁺ and CD8⁺ T lymphocytes were identified. Fluorescence minus one (FMO) controls was used for optimal gating.