Igd bv421

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.

Peyer’s patches (PP) were isolated from the small intestine and cells were extracted as previously described^{16 (link)}. Briefly, PP were finely cut and incubated for 40 min at room temperature with collagenase/DNase. Cells were then filtered through a 70 µm cell strainer and pelleted by centrifuging at 300×g, 4 °C during 5 min and then resuspended in FACS buffer (PBS, 2% FCS, 5 mM EDTA) and counted before antibody staining. For flow cytometry, cells were first incubated on ice in FACS buffer containing an anti-CD16/CD32 antibody to block the Fc receptor for 10 min and then incubated 30 min on ice in the dark with antibodies against the following surface markers : CD45-PE-eF610 (eBiosciences, clone 30-F11), CD19-PerCp-Cy5.5 (Biolegend, clone 6D5), CD3-PeCy7 (eBiosciences, clone 145-2C11), B220-AF647 (BD Biosciences, clone RA3-6B2), IgD-BV421 (BD Biosciences, clone 11-26c.2a), IgM-FITC (BD Bioscience, clone II/41) and IgA-PE (eBiosciences, clone mA-6E1). Cell viability was evaluated using Fixable Viability Dye eFluor 506 (eBiosciences). Cell acquisition was performed using a CytoFlex (Beckman Coulter) and data were analyzed with the CytoExpert software (Beckman Coulter).