Discussion

This report describes a standardized and reproducible procedure for the purification of splenic B cells from mice. The isolated cell population is composed of approximately 96% B cells defined by the expression of B220 and concomitant absence of CD43, Mac-1, and CD3. Of these, more than 80% can be further characterized as resting, mature B2 cells. Cells isolated in this manner maintain their viability over short-term culture in defined serum-free medium and demonstrate reproducible functional responses to ligands for cell-surface receptors.

The suitability of this population as a model for cellular signaling studies is supported by consistent and reproducible responses to several stimuli. A limitation of our approach, however, is that the cell population is not completely homogeneous. A similar limitation would apply to virtually any type of primary cell obtained from an animal. Fortunately, the heterogeneity of the B-cell population is not expected to affect the signaling responses measured by most assays. As we develop and refine our analyses in future experiments, it will be possible to address signaling differences between specific subpopulations (or even stochastic differences between individual cells of a population that appear genuinely homogeneous). For now, this population of cells is suitable for achieving our initial goal - that of identifying ligands that induce interesting signaling responses in B cells.