Anti cd20 clone l27

Cells were first washed with PBS and stained with LIVE/DEAD fixable near-IR (dead cell stain kit, Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature in the dark. Then, cells were washed with PBS supplemented with 1% bovine serum albumin. Extracellular staining was performed by incubating the cells for 30 min at room temperature in the dark with the following antibodies: Anti-CD19 (clone SJ25-C1, BD Bioscience, Franklin Lakes, NJ, USA), anti-CD20 (clone L27, BD Bioscience, Franklin Lakes, NJ, USA), anti-CD27 (clone L128, BD Bioscience, Franklin Lakes, NJ, USA; clone O323, eBioscience, San Diego, CA, USA), anti-CD38 (clone HB7, BD Bioscience, Franklin Lakes, NJ, USA) and anti-IgG (clone G18-145, BD Bioscience, Franklin Lakes, NJ, USA or clone MH16-1, Sanquin Reagents, Amsterdam, the Netherlands). Samples were measured on a FACSCanto, FACSLSRII or FACSLSR Fortessa (BD Bioscience, Franklin Lakes, NJ, USA) and analyzed using FlowJo software version 10 (Treestar, Ashland, OR, USA).

Blood samples were taken from animals undergoing XHALT before and after implantation, in order to monitor effectiveness of immunosuppression and potentially prevent graft rejection. Analysis was performed using flow cytometry. The following antibodies were used to stain peripheral blood: anti-CD3 (clone SP34-2), (BD Pharmingen) anti-CD20 (clone L27), (BD Biosciences). All samples were collected on a FACSCalibur (becton Dickinson, Mountain View, CA) and analysis performed with the FlowJo (TreeStart, Ashland, OR).