Free-flow electrophoresis in subcellular fractionation of human neutrophils

Human neutrophils are endowed with secretory vesicles, an intracellular res ervoir of integral membrane proteins. Secretory vesicles fuse readily with the plasma membrane upon stimulation of the neutrophil, resulting in prompt transportation of various receptors and adhesion proteins to the neutrophi l surface. This upregulation of membrane proteins has been shown to be cruc ial during the sequential steps preceding neutrophil extravasation, However , the lack of separation of secretory vesicles from the plasma membrane whe n the postnuclear supernatant from cavitated neutrophils is centrifuged on a Percoll density gradient has been an obstacle for the investigation of se cretory vesicles. By use of Free Flow Electrophoresis (FFE) we were able to obtain a separation of secretory vesicles from the plasma membrane vesicle s, and this procedure has been a valuable tool in the investigation of secr etory vesicles. Methodological considerations and results obtained by FFE o f light membranes from human neutrophils are presented in this paper. (C) 1 999 Elsevier Science B.V. All rights reserved.