Free-flow electrophoresis in subcellular fractionation of human neutrophils

Human neutrophils are endowed with secretory vesicles, an intracellular reservoir 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 neutrophil surface. This upregulation of membrane proteins has been shown to be crucial during the sequential steps preceding neutrophil extravasation. However, the lack of separation of secretory vesicles from the plasma membrane when the postnuclear supernatant from cavitated neutrophils is centrifuged on a Percoll density gradient has been an obstacle for the investigation of secretory vesicles. By use of Free Flow Electrophoresis (FFE) we were able to obtain a separation of secretory vesicles from the plasma membrane vesicles, and this procedure has been a valuable tool in the investigation of secretory vesicles. Methodological considerations and results obtained by FFE of light membranes from human neutrophils are presented in this paper.