Fortin Carl, Fülöp Tamas
Department of Medicine, Duke University Medical Center, Durham, NC, USA.
Research Center on Aging, Department of Medicine, Immunology Postgraduate Programme, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001-12e Ave Nord, Sherbrooke, QC, Canada, J1H 5N4.
Methods Mol Biol. 2015;1343:1-7. doi: 10.1007/978-1-4939-2963-4_1.
Neutrophils are present within minutes to the site of aggression in the body making them one of the first cells of the immune system to be in contact with incoming threats. The cell functions of neutrophils are elicited through the engagement of surface receptors, some of which are located in a specific region of the membrane called lipid rafts, a functionally segregated region of the membrane enriched with cholesterol and distinct species of sphingomyelin and glycerophospholipids. Lipid rafts are relatively resistant to detergent extraction and this can be taken advantage of to isolate them from the rest of the cell membrane. This chapter will describe a reliable method to obtain lipid rafts from detergent-resistant membrane fractions of human neutrophils. Cells are lysed in an HEPES solution containing 0.5% Triton X-100, supernatants are mixed with a 42% sucrose solution, which is then overlaid with a 35% and 5% sucrose solution. The gradient is centrifuged for 16 h and the resulting fractions can be further analyzed by immunoblotting or subjected to immunoprecipitation.
中性粒细胞在几分钟内就会出现在身体遭受侵袭的部位，这使它们成为免疫系统中最早接触外来威胁的细胞之一。中性粒细胞的细胞功能是通过表面受体的结合来引发的，其中一些受体位于细胞膜的一个特定区域，称为脂筏，这是细胞膜中一个功能上隔离的区域，富含胆固醇以及不同种类的鞘磷脂和甘油磷脂。脂筏对去污剂提取具有相对抗性，可以利用这一点将它们与细胞膜的其余部分分离。本章将描述一种从人中性粒细胞的去污剂抗性膜组分中获取脂筏的可靠方法。细胞在含有0.5% Triton X-100的HEPES溶液中裂解，上清液与42%的蔗糖溶液混合，然后在其上面覆盖35%和5%的蔗糖溶液。将该梯度离心16小时，所得组分可通过免疫印迹进一步分析或进行免疫沉淀。
