title: Isolation of Lipid Rafts from Human Neutrophils by Density Gradient Centrifugation.
abstract: 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.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC, USA. forc2506@gmail.com.
authors.forename: Carl
authors.initials: C
authors.lastname: Fortin
authors.affiliation: 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. Tamas.Fulop@usherbrooke.ca.
authors.forename: Tamas
authors.initials: T
authors.lastname: Fülöp
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry*; D009504:Neutrophils / Q000737:chemistry*
keywords: Detergent-resistant membrane; Flotillin; Human; Lipid raft; Neutrophils; Sucrose