Anti cd20percp

Flow cytometry was performed using a FACSCalibur Cytometer (BD Biosciences); a phenotyping kit (MSC phenotyping kit, Miltenyi) was used to characterize the tMSCs. The following antibodies were used: anti-CD34PerCP, anti-CD45PerCP, anti-CD20PerCP, anti-CD14PerCP, anti-CD73APC, anti-CD9FITC, and anti-CD105PE (BD Pharmingen). Matched isotype controls were applied to determine background fluorescence levels.

Direct immunofluorescence with specific antibodies was performed either on peripheral blood as previously described with some modifications using the following antibodies: anti-IgG1 FITC, anti-CD34-FITC, anti-IgG1-PE, anti-CD3-FITC/CD16+CD56-PE, anti-CD11C-PE, anti-CD21-PE, anti-CD20-PerCP and anti-HLA-DR-PerCP (BD Bioscience, San Jose, CA), anti-CD19-FITC, anti-CD3-APC (Invitrogen, Carlsbad, CA, anti-Lineage-FITC (anti CD3/CD14/CD16/CD19/CD20/CD56), anti-CD45-APC (Biolegend, San Diego, CA, USA) and anti-BDCA2-APC (Miltenyi Biotech, San Diego, CA, USA) [13 (link),21 (link)]. Briefly, after washing the whole blood with PBS, 100ul of blood was stained with the relevant cocktail of antibodies at 4 °C for 30 min followed by red blood cell lysis using BD FACS lysis solution (BD Bioscience, San Jose, CA, USA). Samples were acquired on Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FCS express software (De Novo software, Glendale, CA, USA). During acquisition, a lymphocyte gate was assigned, and 10,000 events were collected for the T cells and NK cells. For DCs, a million ungated events were acquired.