Cd4 percp vit4

Flow cytometric analysis of human TEC was performed by multi-color staining and using the following mAbs: anti-CD326 (EpCam) PE-Vio770 (clone HEA-125), anti-CD45 APC (5B1), anti-HLA-DR, DQ, DP PE (all from Miltenyi Biotec), anti-Ulex-1 FITC (FL 1061, Vector Laboratories, Burlingame, California, USA).
Thymocytes were characterized using a multi-color staining. To discriminate DN, DP, SP4 and SP8 thymocyte subsets, we performed a three-color staining using the following mAbs: CD8 Vio-Blue (clone BW135/80), CD45 APC (5B1) and CD4 PerCP (VIT4) (all mAbs are from Miltenyi Biotec).
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (density: 1.077 g/ml; STEM CELL Technologies, Vancouver, Canada). To isolate total T cells we used the anti-human Pan T cells isolation kit (Miltenyi Biotec). To discriminate CD4⁺ and CD8⁺ T cell subsets we performed a four-color staining using the following mAbs: CD45 APC (clone 5B1), CD3 VioGreen (REA613), CD4 PE-Vio770 (M-T321) and CD8 PE (BW135/80) (all from Miltenyi Biotec).
Surface stainings were performed in PBS with 2% FBS and 0,1% sodium azide for 20 min at 4°C. Cells were acquired using a FACS CantoII (BD Biosciences, San Jose, CA, USA) and analyzed with Flow Jo Software (FLOWJO, LLC, Ashland, OR, USA).

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)].