Activation of mouse lymphocytes by anti-immunoglobulin. III. Analysis of responding B lymphocytes by flow cytometry and cell sorting.

The fluorescence-activated cell sorter (FACS) was used to separate mouse spleen cells that respond by proliferation to stimulation in vitro with anti-µ or anti-κ antibodies. The proliferative response of subpopulations of B lymphocytes bearing various amounts of total membrane (m) immunoglobulin (Ig), mlgM and mlgD, to stimulation by anti-µ, anti-κ, and lipopolysaccharide (LPS) was determined by labeling splenic B lymphocytes with fluoresceinated (Fl) anti-lg, Fl anti-µ, or Fl anti-δ and using the FACS to separate cells into populations bearing various amounts of the fluoresceinated antibody. When lg+ spleen cells were separated on the basis of the total amount of mlg they possessed, those cells with the largest amount of total mlg gave the greatest response to both anti-µ and anti-κ. In contrast, when normal spleen cells were separated on the basis of staining with a Fl anti-µ reagent, the dullest-staining cells exhibited the greatest degree of responsiveness. Separation of cells expressing variable amounts of IgD led to the conclusion that the intermediate-to-bright staining cell population was predominantly responsive to both anti-µ and anti-κ. In general, LPS responsiveness of each of these populations appeared quite similar, except that cells that stained brightly with Fl anti-µ responded better than dull-staining cells. These results suggest that the cells responding to anti-µ and anti-κ possess large amounts of mlg and express a high ratio of surface IgD to IgM. These characteristics are consistent with predictions concerning the phenotype of anti-lg responsive cells based on studies of mlg of lymphocytes from mice with the xid defect and of Lyb 5− B lymphocytes.