Igm fitc

At various points along the time course, cells were collected onto glass slides by cytospin centrifugation and after fixation for 10 min in 95% ethanol, slides were incubated for 10 min in permeabilization buffer (PBS plus 0.2% Triton X-100; Sigma-Aldrich, St. Louis, MO, USA), rinsed briefly in PBS, and stained for cytoplasmic Ig for 45 min at 37 °C in a humidified chamber. After washing with permeabilization buffer and air drying, the slides were cover slipped with Vectashield mounting media with or without DAPI (Vector Labs, Burlingame, CA, USA). Various combinations of the following anti-human Ig antibodies were used at a 1:100 dilution: IgA-TRITC; kappa-FITC; IgM-FITC; lambda-TRITC (all from Southern Biotech, Birmingham, AL, USA); and IgG-AMCA (Vector Labs, Burlingame, CA, USA). Freshly isolated B cells (D0) and D10 IVPCs were collected onto glass slides by cytospin centrifugation and stained for standard morphological features using Wright stain (Thermo Fisher Scientific, Waltham, MA, USA). D0 B cells and D10 IVPCs were analyzed by flow cytometry using a monoclonal PE antibody to SLAMF7/CD319 (Biolegend, San Diego, CA, USA) with an appropriate isotype matched control antibody (Biolegend, San Diego, CA, USA).