title: An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.
abstract: A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on 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 reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-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-beta-cyclodextrin plasma membrane extraction properties.
authors.affiliation: Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Ludmila
authors.initials: L
authors.lastname: Bezrukov
authors.forename: Paul S
authors.initials: PS
authors.lastname: Blank
authors.forename: Ivan V
authors.initials: IV
authors.lastname: Polozov
authors.forename: Joshua
authors.initials: J
authors.lastname: Zimmerberg
mesh_terms: D000327:Adsorption; D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000032:analysis* / Q000378:metabolism; D002999:Clone Cells; D020099:Coated Materials, Biocompatible / Q000378:metabolism; D016895:Culture Media, Serum-Free; D005347:Fibroblasts / Q000378:metabolism*; D005898:Glass / Q000737:chemistry; D051379:Mice; D041681:NIH 3T3 Cells; D015388:Organelles / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis / Q000378:metabolism; D011107:Polylysine / Q000378:metabolism; D013499:Surface Properties; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D047392:beta-Cyclodextrins / Q000378:metabolism / Q000494:pharmacology
chemical_list: D020099:Coated Materials, Biocompatible; D016895:Culture Media, Serum-Free; D010743:Phospholipids; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D011107:Polylysine; D002784:Cholesterol