Quantitative Examination of Changes in Fluidity and Cholesterol Content in Bovine Aortic Endothelial Cells using Phasor Analysis of Laurdan Fluorescence Lifetime

Abstract

Cholesterol is important for maintaining cell membrane integrity and is known to have an effect on the function of ion channels in endothelial cells. Here, we use the properties of Laurdan to investigate how various cellular cholesterol manipulations, altering membrane fluidity and adding a cholesterol analog affect physical properties of bovine aortic endothelial cells (BAEC). Laurdan, a solvatochromic dye, has a spectral red shift in its emission when in a more liquid disordered (Ld) phase compared to a liquid ordered phase (LO). BAECs labeled with Laurdan were treated with methyl-ß-cyclodextrin (MßCD) to remove cholesterol, MßCD saturated with cholesterol to enrich cholesterol content, the cholesterol analog epicholesterol, or fluidizing alcohols benzyl alcohol (BA) or dodecanol and compared to BAECs treated with PBS+Ca2+ as a control. Two-photon excitation was used to excite Laurdan and lifetime measurements were compared at two emission channels (‘blue channel’ and ‘green channel’) in order to differentiate between changes in membrane fluidity and cholesterol content using two methods: traditional phasor analysis and a multiparametric distance analysis using phasor data. Treatment with MßCD, BA and dodecanol resulted in a shift to shorter lifetimes in the blue channel, indicative a more polar (Ld) environment, while enriching cells with cholesterol shifted phasors to longer lifetimes. In the green channel, the phasors of cholesterol enriched cells were shifted in the direction that corresponds to an increase in dipolar relaxation. Only treatment with a higher concentration of MßCD caused a shift in the opposite direction. Using Laurdan and phasor analysis to distinguish changes in fluidity from changes in cholesterol gives us the ability to manipulate these parameters to assist in determining the mechanisms by which cholesterol can impair cell signaling processes.