Purification of human granulocytes by centrifugal elutriation and measurement of transmembrane potential

Abstract

Viable cell samples containing 93% pure granulocytes were obtained from human blood using the techniques of dextran sedimentation followed by centrifugal elutriation. The resting transmembrane potential (E_m) of human granulocytes was estimated using the fluorescent lipophilic cation, Di-S-C₃(5), from the null point for potassium—i.e., the external K concentration at which there is no change in E_m in response to valinomycin (a K ionophore). The E_m of human granulocytes, as calculated from the Nernst potential for K at the null point, is approximately – 100 mV. Data indicate that this large transmembrane potential is due in part to the presence of an electrogenic Na-K pump in human granulocytes which is stimulated by external potassium and inhibited by ouabain.