Isolation of rat peritoneal mononuclear and polymorphonuclear leucocytes on discontinuous gradients of Nycodenz

Abstract

Centrifugation of rat leucocytes from thioglycollate-induced inflammatory peritoneal exudate on a discontinuous gradient of Nycodenz with a density of 1106 g/l and an osmolarity of 400 mosmol/1 separated the polymorphonuclear from the mononuclear leucocytes. The cells on the interphase between the buffer and the gradient medium contained 96% mononuclear leucocytes with a recovery of >60%, and the bottom fraction consisted of 98% polymorphonuclear leucocytes with a yield of 91%, when an exudate isolated 20 h after the injection of thioglycollate was fractionated. Leucocytes isolated from non-inflamed rat peritoneum could be enriched in the fraction of nonspecific esterase-positive cells from 86% to 96% with a recovery of 82% on a gradient with a density of 1091 g/1 and an osmolarity of 325 mosmol/1. The viability of the isolated cells was > 95% (trypan blue exclusion test),and there was no measurable reduction in the fraction of phagocytosing cells (latex and opsonized zymosan) after exposure to the hypertonic gradient material.