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.
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Acknowledgments
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (No. 106634 and No. 106701), the Université de Sherbrooke, and the Research Center on Aging.
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Fortin, C., Fülöp, T. (2015). Isolation of Lipid Rafts from Human Neutrophils by Density Gradient Centrifugation. In: Shaw, A. (eds) Immunosenescence. Methods in Molecular Biology, vol 1343. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2963-4_1
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DOI: https://doi.org/10.1007/978-1-4939-2963-4_1
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2962-7
Online ISBN: 978-1-4939-2963-4
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