Cd27 apc and cd24 apc efluor780

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.

Flow cytometry was used to follow changes in classical B cell immunophenotype markers (CD19, IgD, IgM, and CD27) as well as those involved in energy pathways including CD24, CD39, CD73, and CD38 throughout in vitro culture. B cell numbers, B cell growth (total mass), and viability were also calculated using flow cytometry. Briefly, PBMCs or B cells were stained for 20 min with fluorescent conjugates of monoclonal antibodies to CD19-Alexa Fluor 700, IgD-BV421, and IgM-BV605 (BD Biosciences, San Jose, CA, USA), CD27-APC and CD24-APC eFluor780 (eBioscience, San Diego, CA, USA), CD39-FITC, CD73-PE, and CD38-PerCP.Cy5.5 (Biolegend, San Diego, CA, USA), and a viability marker (LIVE/DEAD™ Fixable aqua dead cell stain, ThermoFisher Scientific, Waltham, MA, USA). The cells were washed and resuspended in PBS and acquired within 24 h on a BD LSR FortessaTMX-20. Total mass was calculated by combining the mass of debris, dead cells, and live cells in each culture well using the following formula: (FSC × mean number viable + FSC × mean number dead + FSC × mean number debris). Compensation beads (BD, Biosciences, San Jose, CA, USA) were used to optimize the fluorescence compensation settings for multicolor flow cytometric analysis. A minimum of 100,000 events in the lymphocyte gate was collected.