Abstract
Plasma membrane lipid rafts are highly ordered membrane microdomains enriched for glycosphingolipids and cholesterol, which play an important role during T-cell antigen receptor (TCR) signaling. Our previous work has demonstrated that plasma membrane lipid composition is an important determinant of human CD4+ T-cell function and that defects in lipid raft expression contribute to CD4+ dysfunction in patients with autoimmunity. In this chapter we share three flow cytometry-based methods to quantitatively analyze plasma membrane lipid composition in primary human CD4+ T cells. We describe the quantification of glycosphingolipid expression using cholera toxin subunit B, cholesterol expression using filipin staining, and membrane “lipid order” using di-4-ANEPPDHQ. These methods can easily be adapted to analyze different cell types.
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Acknowledgments
Work from KEW was funded by a British Heart Foundation PhD Studentship (FS/13/59/30649). Work from ECJ was funded by Arthritis Research UK Fellowships (20085 and 18106), Lupus UK, The Rosetrees Trust (M409), and University College London Hospital Clinical Research and Development Committee project grant (GCT/2008/EJ and Fast Track grant F193).
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Waddington, K.E., Pineda-Torra, I., Jury, E.C. (2019). Analyzing T-Cell Plasma Membrane Lipids by Flow Cytometry. In: Gage, M., Pineda-Torra, I. (eds) Lipid-Activated Nuclear Receptors. Methods in Molecular Biology, vol 1951. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9130-3_16
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DOI: https://doi.org/10.1007/978-1-4939-9130-3_16
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