Cd27 clone o323

PBMCs were stained with antibodies specific for CD19 (clone HIB19, eBiosciences, San Diego, CA), IgM (clone MHM-88), IgD (clone IA6-2), and CD27 (clone O323, all from BioLegend, San Diego, CA). B cell populations were sorted using an Aria II (BD Biosciences) into naive (CD19+CD27- and IgM+ and/or IgD+) or memory (CD19+CD27+) cells. Purity of sorted populations was tested and confirmed to have <2% contamination by the other B cell subset. Cells were lysed using TRIzol Reagent (Life Technologies, Grand Island, NY) and stored at -80°C until RNA extraction.

B cells were Fc blocked (BD Biosciences, San Jose, CA) prior to staining. Cells were analyzed using an LSR Fortessa cytometer (BD Biosciences), with gating performed by first setting a wide lymphocyte gate by forward and side scatter, followed by two sets of doublet exclusion, first by FSC-A by FSC-H and then by FSC-A by FSC-W, and by excluding dead cells with a viability stain (Life Technologies, Grand Island, NY). B cell purity was determined during isolation and at baseline by staining for CD19 (clone HIB19, eBiosciences, San Diego, CA). Cell phenotype was determined by staining IgM (clone MHM-88), CD27 (clone O323), and CD38 (clone HIT2, all from BioLegend, San Diego, CA), with results reported for the live cells gate. B cell proliferation was determined by dilution of CFSE dye, with results reported for the live singlet lymphocyte gate.