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

doi:10.1016/j.ab.2009.07.027

Analytical Biochemistry

Volume 394, Issue 2, 15 November 2009, Pages 171-176

https://doi.org/10.1016/j.ab.2009.07.027 Get rights and content

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-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.

Section snippets

Chemicals and solutions

Poly-l-lysine solution (Sigma Chemical, St. Louis, MO, USA), boric acid (Mallinckrodt Baker, Paris, KY, USA), Tris-buffered saline (TBS, 10×, Cellgro, Mediatech, Herndon, VA, USA), ultrapure water (KD Medical, Columbia, MD, USA), and Pyrex dishes, 100 × 20 mm, (Corning, Corning, NY, USA) were used. Fluorescent probes for staining mitochondria (MitoTracker Green FM [MTG]), lysosomes (LysoTracker Red DND-99 [LTR]), Golgi apparatus (BODIPY FL C5-ceramide), endoplasmic reticulum (Rhodamine B, hexyl

Results

The purity of the plasma membrane preparation was evaluated by labeling with fluorescent, organelle-specific probes. An example of HAB2 cells and plasma membranes of HAB2 cells labeled with BODYPY FL C5-ceramide specific for the Golgi apparatus is shown in Fig. 2. The detection of nuclei, endoplasmic reticulum, lysosomal membrane, Golgi, and mitochondria in plasma membrane preparations corresponded to contamination levels, relative to intact cells, of 0.9, 2.9, 3.4, 5.7, and 3.0%, respectively,

Discussion

Polylysine-coated glass plates can be used to obtain quantities of plasma membrane suitable for both biochemical and microscopy studies. We used our method of plasma membrane preparation to study how MβCD treatment extracts cholesterol from both plasma membranes and intact HAB2 cells. MβCD treatment extracts cholesterol from intact cells and their membranes in a temperature-dependent way. Nearly complete removal of plasma membrane cholesterol can be achieved by extraction at physiological

Acknowledgments

The authors thank K. Melikov, E. Zaitseva, and J. Mazar for fruitful discussions and suggestions. This study was supported by the Intramural Research Program of the National Institutes of Health, Eunice Kennedy Shriver National Institute of Health and Human Development.

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Published by Elsevier Inc.

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

Abstract

Section snippets

Chemicals and solutions

Results

Discussion

Acknowledgments

Plasma membranes of mammalian cells

J. Cell Biol.

Purification and properties of HeLa cell plasma membranes

J. Biol. Chem.

Isolation of plasma membrane fragments from HeLa cells

J. Cell Biol.

Cellular membranes: the isolation and characterization of the plasma and smooth membranes of HeLa cells

Arch. Biochem. Biophys.

HeLa cell plasma membranes: I. 5-Nucleotidase and ouabain-sensitive ATPase as markers for plasma membranes

J. Cell Biol.

Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells

J. Histochem. Cytochem.

Observing FceRI signaling from the inside of the Mast cell membrane

J. Cell Biol.

Plasma membrane: rapid isolation and exposure of the cytoplasmic surface by use of positively charged beads

Science

Membrane isolation on polylysine-coated beads: plasma membrane from HeLa cells

J. Cell Biol.

Membrane isolation on polylysine-coated glass beads: asymmetry of bound membrane

Biochim. Biophys. Acta

Retention of lipid asymmetry in membranes on polylysine-coated polyacrylamide beads

Biochim. Biophys. Acta

Coupling polylysine to glass beads for plasma membrane isolation

Biochim. Biophys. Acta

Transbilayer mapping of membrane proteins using membranes isolated on polylysine-coated polyacrylamide beads

Biochim. Biophys. Acta