Purification and Characterization of Mouse Splenic B Lymphocytes

Abstract

Initial efforts of Alliance for Cellular Signaling (AfCS) laboratories have focused on establishing standard and reproducible procedures for purifying B lymphocytes (B cells) from mouse spleen. Our goal was to obtain cultures that permit reliable measurement of short-term signaling events and changes in gene expression. This report describes a negative-selection procedure for B-cell enrichment and characterization of the isolated cell population using multiparameter fluorescence-activated cell sorting (FACS) analysis. Results show that we obtain approximately 47 x 10 6 cells per spleen, of which 96% contain the B220 marker typically expressed on B cells. More than 80% of these cells can be further characterized as resting, mature B2 cells. Conditions for maintaining viable cells in serum-free medium for short-term culture were defined. Supplemented Iscove’s Modified Dulbecco’s Medium (SIMDM) was most consistent among several media in maintaining cell viability for 24 hours. B cells also showed consistent and reproducible signaling responses to various stimuli. The cell preparations contain few non-B-cell contaminants and exhibit expected heterogeneity in the expression of some cell-surface markers. The procedure described is conveniently transferable and provides a suitable model for the study of global signaling responses in primary cells.