Cd39 fitc

Specific antibodies used, were targeting: P2X7 (APR-008, Alomone); P2X4 (APR-002, Alomone); PDHX (H-130, Santa Cruz); p-p38 Thr180/Tyr182 (28B10, Cell Signalling Technologies); E-selectin (NBP1-45545, Novus Biologicals); CD31-AlexaFluor488 (Clone Mec13.3, Biolegend), and CD39-FITC (Clone A1, Biolegend). HRP-conjugated secondary antibodies were from Dako. AlexaFluor conjugated antibodies, TO-PRO-3 and aqueous mounting media (Prolong Gold Antifade Mountant) were from Invitrogen. All other reagents were from Sigma Aldrich unless specified.

PBMCs or B cells were stained for 20 min with fluorescent conjugates of CD19-Alexa Fluor 700, CD38-PerCP.Cy5.5, CD39-FITC and CD73-PE (Biolegend, San Diego, CA) IgD-BV421, IgM-BV605 (BD Biosciences, San Jose, CA) CD27−APC and CD24-APC eFluor780 (eBioscience, San Diego, CA), and a viability marker (LIVE/DEAD^TM Fixable aqua dead cell stain, ThermoFisher Scientific, Waltham, MA). Cells were washed (centrifuged for 5 min 300 × g at room temperature) and resuspended in PBS and acquired within 24 h on a BD LSR Fortessa^TMX-20. Compensation beads (BD, Biosciences, San Jose, CA) were used to optimize fluorescence compensation settings for multicolour flow cytometric analysis. A minimum of 100,000 events in the lymphocyte gate was collected. Naïve and Memory B cell subsets were defined as in our previous publication (16 (link)) based on the classification described in Ref (20 (link)). Representative plots of the classical B-cell subsets defined by IgD/CD27 and IgD/CD38 using this system are shown in Supplementary Figure 1.