Complement action on secretory cells identified by the reverse hemolytic plaque assay: modified assay eliminates...

The application of a hemolytic plaque assay to antigen-secreting endocrine cells has brought about great advances in the study of regulation of hormone secretion. The reverse hemolytic plaque assay (RHPA) has enabled quantitation of secretion at the single cell level with simultaneous analysis of the population response. Moreover it has allowed unambiguous identification of specific cell types in mixed cell populations while maintaining the viability of the cells for further physiological experiments. Concern has arisen, however, regarding potential complement attack on those cells of interest, causing sublytic permeabilization leading to altered physiological function. To test this possibility, prolactin release from dispersed anterior pituitary cells was quantitated in two protocols of the RHPA. Cells were exposed to complement either subsequent to the termination of antiserum incubation or simultaneously with antiserum incubation, during which time hormone release is being detected. The presence of complement during antiserum incubation resulted in significant increases in mean plaque area as compared to the separate incubation procedure (13 709 ± 698vs 9251 ± 547 µm(2)). Analysis of the population profile of plaques indicated that the increased mean plaque area reflected a rightward shift in the frequency distribution of plaque size. The general increase in hormone release in the antibody/complement group is consistent with a predicted permeabilizing action of the complement on the secretory cells. To avoid 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 unequivocally identified without being exposed to complement.