Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll

Abstract

Rat mast cells from peritoneal washings were purified by centrifugation on a medium containing silica particles coated with PVP (Percoll). By isopycnic centrifugation on continuous Percoll gradients, the buoyant density of mast cells ranged from 1.09 to 1.17 and was consistently lower in mast cells from younger rats than in mast cells from older rats. In suspensions uncontaminated with blood, the mast cells could be isolated by a simple centrifugation procedure based on density differences (step I). Contaminating red blood cells were removed by a second short centrifugation of velocity gradient type, using a linear Percoll gradient of low density. The mast cell purity was 91-98% and the recovery 63-100%. Purified mast cells reacted normally in a viability test employing fluorescein diacetate, retained amines and heparin, and reacted normally with amine release following incubation with Polymyxin B or antigen (immunised rats). Electron microscopy revealed excellent ultrastructural preservation of the mast cells and infrequent uptake of Percoll particles by endocytosis.