Cd22 apc

Peripheral blood mononuclear cells (PBMCs) were isolated, frozen and thawed as described before [24 (link)]. PBMCs were washed twice in PBS, followed by a 15-minute incubation at 37°C with eFl670 (eBioscience). Next, PBMCs were cultured in the presence or absence of 500 ng/mL CpG (Hycult Biotech, Uden, the Netherlands), with 3 μM MPA or 3 μM 6-MP (Sigma-Aldrich, St. Louis, MO, USA). After 3 days of culture, samples were washed and stained with anti-human CD19-eFluor 450, CD22-APC (BioLegend, San Diego, CA, USA) and CD3-BV786 (BD Biosciences). At least 200.000 cells were acquired and plotted using Kaluza v1.5a or FlowJo v10. In S2 Fig and Fig 2A representative gating examples are given.

FACS analysis of cell surface CAR and protein expression was performed using a CytoFLEX S flow cytometer (Beckmam Coulter, CA, USA). CD22-CAR was detected by incubation with APC-F(ab)2 (Jackson Immunoresearch, USA). The following human monoclonal antibodies were used for detection of cell surface proteins: CD22-APC, CD22-PE, CD19-PE, CD45-PerCP/Cy5.5, CD3-APC/Cy7, CD8-Pacific Blue, CD4-APC, CD45RO-PE/Cy7, CD45RA-FITC, CCR7-APC (all from BioLegend). CD22 site density was quantified by Quantity-PE beads (BD Biosciences, USA) following instructions. Both GFP-expressing cells and CFSE dye-labeled cells were identified through the FITC channel.