Cd3 fitc hit3a

FACSAriar (BD Bioscience) was used to perform fluorescent expression analysis. Cells were harvested the following days after transfection and stained with mouse anti-human antibody labeled by fluorescence for 30 m in 4 °C in darks as follows: CD3-PerCP-CY5.5(OKT-3, eBioscience) or CD3-FITC (HIT3a, BD Bioscience), CD4-APC (RPA-T4, BD Bioscience), CD8-PE (HIT8a, BD Bioscience), PD-1-PerCP-CY5.5 (EH12.1, BD Bioscience), CD25-PE (BC96, eBioscience), CD62L-FITC (DREG-56,e Bioscience), CD27-PE (0323, eBioscience), CD28-PE (CD28.2, eBioscience), CD45RO-PE (UCHL1, BD Bioscience), CD69-PE (FN50, BD Biolegend), HLA-DR-PE (G46-6, BD Bioscience).

Purity of immune cell subtypes was assessed by labeling purified fractions with the antibodies CD4-PerCp (VIT4; Miltenyi Biotec, Macquarie Park, Australia), CD8-FITC (BW135/80; Miltenyi Biotec, Macquarie Park, Australia), CD20-PE (B lymphocytes; LT20; Miltenyi Biotec, Macquarie Park, Australia), CD14-PE (TUK4) and CD3-FITC (HIT3a; BD Pharmingen, Sparks, MD)/CD56-APC (AF12-7H3; Miltenyi Biotec, Macquarie Park, Australia) for NK cells. Flow cytometry of labeled PBMC and purified cell subsets was performed using a CyAn ADP analyzer (Beckman Coulter) and the data analysed using WEASEL (v3.0). Purified fractions for each of the cell subsets were only used in subsequent analyses if purity was 90% or greater.
In order to assess MERTK expression on the surface of monocytes, PBMCs were isolated from whole blood and monocytes identified using antibodies directed against the markers CD14-PE [TUK4 (Miltenyi Biotec, Macquarie Park, Australia)] and CD16-FITC [VEP13 (Miltenyi Biotec, Macquarie Park, Australia)] as previously described [44 (link)]. Cell surface MERTK protein was detected using human Mer APC-conjugated antibody [Clone #125518 (R&D Systems, Minneapolis, MN)] and compared with the appropriate isotype control [APC-conjugated Mouse IgG1, IS5-21F5 (Miltenyi Biotec, Macquarie Park, Australia)].