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 purchased 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 25 minutes on ice, washed with permeabilization buffer containing saponin (BD biosciences, San Diego, CA), and incubated with the Pacific Orange labeled surface/intracellular marker anti-mouse anti-Ig heavy and light chain (Invitrogen) as described [18 (link)]. Compensation is prepared using flow through sample aliquots individually incubated with the same antibodies used in the staining mixture. The FITC, hapten-PE and the PE-AF647 compensation tubes were stained respectively with CD4-FITC, CD4-PE and CD4-PE-Cy5.
Cells were fixed in formaldehyde (Cytofix/Cytoperm, BD biosciences, San Diego, CA) for 25 minutes on ice, washed with permeabilization buffer containing saponin (BD biosciences, San Diego, CA), and incubated with the Pacific Orange labeled surface/intracellular marker anti-mouse anti-Ig heavy and light chain (Invitrogen) as described [18 (link)]. Compensation is prepared using flow through sample aliquots individually incubated with the same antibodies used in the staining mixture. The FITC, hapten-PE and the PE-AF647 compensation tubes were stained respectively with CD4-FITC, CD4-PE and CD4-PE-Cy5.