Multi-Marker Flow Cytometry Immunophenotyping

Cell suspensions were incubated, for 25 minutes on ice, with fluorochrome labeled anti-mouse antibodies for the following B cell surface markers: FITC anti-GL7, PE-Cy7 anti-B220, APC anti-IgM, AF700 anti-CD38, eF450 anti-IgD; and for the following APC-eF780 labeled anti-mouse antibodies for non-B cell surface markers: anti-CD90.2, anti-CD11c, anti-Ly-6G and anti-F4/80. All antibodies were from eBioscience (San Diego, CA) with the exception of FITC anti-GL7 (BD Pharmingen). Cells were fixed in formaldehyde (Cytofix/Cytoperm, BD biosciences, San Diego, CA) for 30 minutes on ice, washed with permeabilization buffer (BD biosciences, San Diego, CA), and incubated with Pacific Orange labeled surface/ intracellular marker anti-mouse anti-Ig heavy and light chain as described [17] (link), [20] . Compensation was prepared for each individual fluorochrome-coupled antibody used in the staining mixture.