Cd45 apc clone 5b1

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).

Following the MINDEC procedure, enriched samples were resuspended in 100 μL cold staining buffer along with 25 μL FcR blocking reagent (Miltenyi Biotech). The samples were further processed following one of the following three procedures. Samples from recovery experiments to be enumerated by flow cytometry were incubated with 2.5 μL EpCAM-FITC (clone HEA-125, Miltenyi Biotech), 2.5 μL MCAM-FITC (clone OJ79c, AbD Serotec^®; for cell line SDM103T2 only), and 2.5 μL CD45-APC (clone 5B1, Miltenyi Biotech). Samples from linearity experiments to be enumerated by microscopy were incubated with 2 μL EpCAM-FITC (clone HEA-125, Miltenyi Biotech), 2 μL CD45-DyLight550 (clone T29/33, Leinco Technologies, Inc.), and 2 μL Hoechst 33342 (Molecular Probes). Finally, patient samples were incubated with 2 μL EpCAM-FITC (clone HEA-125, Miltenyi Biotech), 2 μL MCAM-FITC (clone OJ79c, AbD Serotec^®), 2 μL CD45-DyLight550 (clone T29/33, Leinco Technologies, Inc.), and 2 μL Hoechst 33342 (Molecular Probes). All samples were incubated for 20 minutes in darkness at room temperature during staining, and were subsequently resuspended in an appropriate volume of cold staining buffer (500 μL for flow cytometry experiments, 50 μL for microscopy enumeration experiments, and 150 μL for patient samples).