Abstract
Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and animal cells and comprehensively analyse their lipid composition.
Keywords: Lipid rafts, Lipids, Lipidomics, Cholesterol, Membrane
Background
Lipid rafts are distinct cholesterol- and sphingomyelin-rich structures in cell membranes, immersed into liquid-disordered surrounding membrane (Lingwood and Simons, 2010). They provide a “solid” platform to spatially organize elements of various signaling pathways as well as exo- and endocytosis machineries. The functional properties of lipid rafts are determined by interaction between raft lipids and proteins. Changes in raft lipid composition, both physiological and pathological, have striking consequences for the activity of the pathways originating from rafts and represent an important and underappreciated layer of their regulation. Isolation of lipid rafts is complicated by their dynamic nature and heterogeneity. Methods of raft isolation based on their resistance to detergents have been consistently criticized (Gaus et al., 2005). Here we describe a detergent-free method for isolating lipid rafts and establishing their lipid composition using comprehensive lipidomics analysis. One example of using this protocol has been recently published (Mukhamedova et al., 2019).
Part I: Isolation of Lipid Rafts The protocol described below was used to isolate rafts from RAW 264.7 murine macrophages, THP-1 human macrophages and SH-SY5Y human neuroblastoma cells, but is applicable to most cultured mammalian cells.
Materials and Reagents
Equipment
Procedure
This protocol is based on technical manuals from Alere Technologies AS, Norway.
Expected Results The example of lipid raft isolation from SH-SY5Y human neuroblastoma cells is shown in Figure 1. In this experiment cellular cholesterol was trace-labelled with [3H]cholesterol (see Low et al., 2012) for labeling methodology) and fractions with maximum concentration of labelled cholesterol were defined as raft fractions (fractions 5-9). An example of using flotillin-1 (membrane protein abundantly present in lipid rafts and considered a raft marker) to identify lipid raft fractions has been recently published and also showed maximum abundance of this marker in fractions 5-9 (Mukhamedova et al., 2019). Figure 1. Representative profile of gradient centrifugation of plasma membranes from SH-SY5Y human neuroblastoma cells
Recipes
Part II: Lipidomics Analysis of Lipid Rafts This lipidomics protocol provides quantification of a large range of lipid species relative to appropriate internal standards. It is recognized that it is not possible to provide stable isotope internal standards for each lipid species and that differences between the response factors of the analytes and the corresponding internal standards are not adjusted for. Thus, the lipidomic measures should be considered as relative, rather than absolute, quantification.
Software
Competing interests
The authors declare no competing interests related to this work.
Acknowledgments
The authors were supported by the grant from the National Institutes of Health (HL131473 to DS). This protocol was modified from previous work: Mukhamedova et al. (2019) and Huynh et al. (2019).
References
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