Purification of adrenal medullary chromaffin cells by density gradient centrifugation

Abstract

A method has been developed for the isolation of chromaffin cells from guinea pig adrenal glands. Crude suspensions of adrenal cells are prepared by the digestion of adrenal glands with collagenase. Chromaffin cells are then purified from these crude suspensions by isopycnic centrifugation through a 5–25% (w/v) gradient of metrizamide. More than 90% of the cells in the preparation are viable chromaffin cells, as judged by trypan blue exclusion and fluorescence histochemistry. A more convenient method for monitoring the purity of chromaffin cell preparations, using neutral red staining, is also described. Chromaffin cells are the primary neutral red staining cell in the adrenal gland, and more than 90% of the purified chromaffin cells stain with this dye. Purified chromaffin cells contain 400 ± 50 nmol of epinephrine/mg protein (133 ± 16 nmol/10 6 cells), and secrete epinephrine in response to acetylcholine. At a concentration of 100 μM, acetylcholine causes a 10- to 20-fold increase in the secretion of epinephrine from the cells. The method described in this paper is a useful procedure for the preparation of pure, functional chromaffin cells.