A SIMPLIFIED METHOD FOR CA2-CELLS THAT USES FLOW-CYTOMETRY( FLUX MEASUREMENT ON ISOLATED HUMAN B)

A method for Ca2+ flux measurement on isolated human peripheral B cell s that uses flow cytometry is described, B cells were isolated by anti -CD19 magnetic bead sorting, and Ca2+ Bur was measured with the fluo-3 reagent on a standard single-laser flow cytometer, The response of B- cell stimulation by anti-immunoglobulin B (anti-IgM), anti-IgD, protei n A, concanavalin A, and ionomycin was determined, Percentage of respo nder B cells, the level of Ca2+, and the time of peak stimulation were measured, Bound anti CD19 monoclonal antibody coupled with small para magnetic particles did not affect Ca2+ flux, All the isolated B cells responded maximally at 10 s with stimulation by 8 mu g of ionomycin, T he average isolated preparation contains 70% IgM(+) and 85% IgD(+) cel ls, all of which showed peak stimulation with 10 mu g of anti-IgM and anti-IgD per mi, respectively, at 30 s, Only at high concentrations of 80 mu g/ml, concanavalin A produced a slower response, peaking at 90 s after stimulation, Stimulation with 20 mu g of protein A per mi resu lted in Ca2+ flux in only 30 to 60% of cells that had a rapid response and maximal stimulation resembling the pattern of activation of ionom ycin, B cells from three patients with mixed cryoglobulinemia,vith hig h concentrations of monoclonal rheumatoid factors showed stimulation w ith aggregated IgG, whereas those from healthy control subjects did no t, demonstrating the applicability of the methodology to detection of specific antigen stimulation of B cells, This methodology may be usefu l in testing the functional capacity of B cells in a variety of diseas es, The methodology may also prove useful in studying antigen-specific B-cell responses when they involve a significant percentage of B cell s.