An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes

摘要

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based upon the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5 %. The method uses inexpensive, commercially available, polylysine and re-usable glass plates. Plasma membrane preparations can be made in 15 minutes. Using this method, we determined that methyl-β-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol:phospholipid ratio from intact cells does not reflect methyl-β-cyclodextrin plasma membrane extraction properties.