Isolated adipocytes: An assessment of cell surface changes during their preparation

Abstract

A method is described, based on the detection of adipocyte-specific cell surface antigens, which allows assessment of the relative surface damage incurred by the cells when they are prepared under a variety of conditions. Using the method it is possible to develop, for any set of reagents, a set of cell isolation conditions (collagenase concentration, time of incubation) which will produce minimally damaged cells which exhibit high levels of specific cell surface immunoreactivity. Under certain conditions a recovery from limited surface damage can be achieved, although, when cells are prepared under more extreme conditions irreversible surface damage occurs. The surface morphology of the cells as revealed by scanning electron microscopy, is also clearly affected by the conditions of cell isolation. The method has been used to define the conditions necessary for the isolation of cells to be used in the study of subtle biochemical responses.

The Heterogeneity of Isolated Adult Rat Leydig Cells Separated on Percoll Density Gradients: An Immunological, Cytochemical, and Functional Analysis

“…As cell damage caused by the dispersal conditions may obscure any preexisting Leydig cell heterogeneity, several modifications of the standard collagenase dispersal procedure were made to reduce proteolytic and mechanical cell damage without greatly reducing Leydig cell yield. This involved addition of serine and thiol protease inhibitors to the collagenase solution to reduce or neutralize the activity of contaminating proteases, which may damage the Leydig cell membrane (15,16); vascular perfusion with collagenase to ensure rapid and uniform access to the interstitial tissue and to reduce the presence of blood cells; and a relatively slow shaking speed during dissociation.…”

The lipoprotein lipase of white adipose tissue. Studies on the intracellular distribution of the adipocyte-associated enzyme

“…Cells were prepared by a modification of the procedure of Rodbell (1964) as described by Cunningham & Robinson (1969) and Cryer et al (1975). To minimize the morphologically and immunologically detectable cellsurface damage (Al-Jafari et al, 1986) produced by proteolytic action during the isolation procedure, crude bacterial collagenase (150 units/mg; from Clostridium histolyticum; Sigma Chemical Co., type II, code C2139) was used at relatively low concentrations (0.5 mg/ml) in the isolation medium. The yield of cells under these conditions was approx.…”

“…The functional significance of the lipoprotein lipase activity detected at the adipocyte plasma membrane is at present only a matter of speculation, but other experiments (Al-Jafari & Cryer, 1986) show that the size of this pool of enzyme molecules alters in a concerted fashion in response to specific extracellular effectors. It would be reasonable to propose that the lipoprotein lipase at this site represented a pool of enzyme molecules which either may continue, via some as yet unknown transport system, en route to the endothelium, or may be subject to re-uptake by the adipocyte.…”