An adhesion-based method for plasma membrane isolation: Evaluating cholesterol extraction from cells and their membranes
An adhesion-based method for plasma membrane isolation: Evaluating cholesterol extraction from cells and their membranes
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DOI：
10.1016/j.ab.2009.07.027
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发表时间：
2009-11-15
影响因子：
2.9
通讯作者：
Zimmerberg, Joshua
中科院分区：
文献类型：
--
作者：
Bezrukov, Ludmila;Blank, Paul S.;Zimmerberg, Joshua
提出了一种从附着细胞中分离大量可直接接触的质膜的方法。该方法是基于细胞与玻璃板上的聚赖氨酸吸附层的粘合，然后用冰冷蒸馏水进行低渗裂解和随后的洗涤步骤。确定了镀膜玻璃板的最佳条件和细胞贴壁时间。没有使用额外的化学或机械处理。细胞器对分离质膜的污染小于5%。该方法使用廉价的、商业上可获得的聚赖氨酸和可重复使用的玻璃板。质膜制备可在15分钟内完成。使用这种方法，我们确定了甲基-β-环糊精从成纤维细胞及其质膜中差异地提取胆固醇，并且这些差异与温度有关。对完整细胞中胆固醇/磷脂比率的测定不能反映甲基-β-环糊精的质膜提取特性。由爱思唯尔公司出版。
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. Published by Elsevier Inc.