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

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.