Cd38 pe texas red

Isolated B cells were washed and stained with Live/Dead viability discrimination dye (Life Tech #L34968). Cells were subsequently blocked in 2% BSA supplemented with Fc Blocker (BioLegend) for 15 min and then stained with antibodies against B cell surface makers for 1 h [all antibodies were from BD Biosciences except CD38-PE/Texas Red (Life Technologies #MHCD3817); CD19-BV510, #562847; CD20-BUV396, #563782; CD27-BV421, #560448; IgD-APC, #348222; IgM-PerCP/Cy5.5, #314512; CD138-PE, #552026; IgG-PE/Cy7, #409316; CD45-APC/Cy7, #368516]. After staining, cells were washed two times in PBS without Mg⁺⁺ or Ca²⁺ and then fixed in 2% PFA before analysis on a BD Fortessa or BD LSR flow cytometer. Plasmablasts were defined as CD19⁺CD20⁺IgD⁻CD27⁺CD38⁺ B cells. B cell activation was determined with CD86 surface staining (#562432). Ig production was determined by intracellular staining with anti-IgA (Life Tech #Z25002), anti-IgE (Biolegend #325510), and anti-IgG antibodies in plasmablasts; AID was detected with antibody #Z25302 (Life Tech). For intracellular IRF5 staining, after overnight fixation, cells were permeabilized the following day in 0.1% Triton X-100 and rinsed in PBS 2× before blocking in 2% BSA solution. IRF5 staining was performed using anti-IRF5 antibody conjugated to Alexa Fluor 488 (Abcam Catalog#: AB193245).