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. 2023:2625:353-364.
doi: 10.1007/978-1-0716-2966-6_30.

C-Laurdan: Membrane Order Visualization of HEK293t Cells by Confocal Microscopy

Sean D Meehan  1   2   3 , Colin Hayter  4 , Sanjoy K Bhattacharya  5   6   7
Affiliations

C-Laurdan: Membrane Order Visualization of HEK293t Cells by Confocal Microscopy

Sean D Meehan et al. Methods Mol Biol. 2023.

Abstract

Membrane order is a biophysical characteristic dependent on cellular lipid makeup. Cells regulate the membrane structure as it affects membrane-bound protein activity levels and membrane stability. Spatial organization of membrane lipids, such as lipid rafts, is a proposed theory that has been indirectly measured through polarity-sensitive fluorescent dyes. C-Laurdan is one such dye that penetrates plasma and internal membranes. C-Laurdan is excited by a single 405 nm photon and emits in two distinct ranges depending on membrane order. Herein, we present a protocol for staining HEK293t cells with C-Laurdan and acquiring ratiometric images using a revised ImageJ macro and confocal microscopy. An example figure is provided depicting the effects of methyl-β-cyclodextrin, known to remove lipid rafts through cholesterol sequestration, on HEK293t cells. Further image analysis can be performed through region of interest (ROI) selection tools.

Keywords: C-Laurdan; Confocal microscopy; HEK293t cells; Membrane order.

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