COMPLEMENT ACTION ON SECRETORY-CELLS IDENTIFIED BY THE REVERSE HEMOLYTIC PLAQUE-ASSAY - MODIFIED ASSAY ELIMINATES EXPOSURE OF SECRETORY-CELLS TO COMPLEMENT

The application of a hemolytic plaque assay to antigen-secreting endoc rine cells has brought about great advances in the study of regulation of hormone secretion. The reverse hemolytic plaque assay (RHPA) has e nabled quantitation of secretion at the single cell level with simulta neous analysis of the population response. Moreover it has allowed una mbiguous identification of specific cell types in mixed cell populatio ns while maintaining the viability of the cells for further physiologi cal experiments. Concern has arisen, however, regarding potential comp lement attack on those cells of interest, causing sublytic permeabiliz ation leading to altered physiological function. To test this possibil ity, prolactin release from dispersed anterior pituitary cells was qua ntitated in two protocols of the RHPA. Cells were exposed to complemen t either subsequent to the termination of antiserum incubation or simu ltaneously with antiserum incubation, during which time hormone releas e is being detected. The presence of complement during antiserum incub ation resulted in significant increases in mean plaque area as compare d to the separate incubation procedure (13 709 +/- 698 vs 9251 +/- 547 mu m(2)). Analysis of the population profile of plaques indicated tha t the increased mean plaque area reflected a rightward shift in the fr equency distribution of plaque size. The general increase in hormone r elease in the antibody/complement group is consistent with a predicted permeabilizing action of the complement on the secretory cells. To av oid this potentially damaging effect of complement on secretory cells to be used in subsequent physiological experiments, we have developed a modification of the RHPA in which the secretory cells are unequivoca lly identified without being exposed to complement.