DETECTION OF HORMONE RELEASE FROM INDIVIDUAL CELLS IN MIXED POPULATIONS USING A REVERSE HEMOLYTIC PLAQUE ASSAY

TLDR

The authors propose using sequential reverse hemolytic plaque assays on identified pituitary cells, coupled with immunocytochemistry, autoradiography or electron microscopy, to determine whether all hormone‑producing cells respond to a secretagogue and whether dual‑hormone cells preferentially release one hormone. The assay employs prolactin‑secreting pituitary cells plated with staphylococcal protein‑A coated ovine erythrocytes, prolactin antiserum, and complement to generate microscopically identifiable hemolytic plaques. Plaques appear within 15–30 min, peak at 2 h, and are formed by about 70 % of adenohypophyseal cells; TRH increases while dopamine decreases plaque size and number early, and the assay can detect secretion of any hormone for which an antibody is available.

Abstract

Prolactin (Prl) secreting cells in a mixed pituitary cell culture form microscopically-identifiable plaques (zones of hemolysis around the lactotropes) when incubated in a monolayer with staphylococcal protein-A-coated ovine erythrocytes in the presence of Prl antiserum and complement. Plaques form first at 15–30 min and are maximal in size and number at 2 h. Approximately 70% of the adenohypophyseal cells form plaques under these conditions. TRH increases, and dopamine decreases, the size and number of plaques at early times during incubation. This reverse hemolytic plaque assay probably can be used to detect any cell secretion for which an antibody is available. This technique, or a modified version of it in which sequential plaque assays are performed on identified cells–used together with immunocytochemistry, autoradiography or electron microscopy of those cells–should provide better answers to commonly asked questions about secretory systems: Do all or only a subset of cells containing the same hormone respond to a particular secretagogue Can cells that contain two hormones release one of them preferentially