The cell membrane is a bilayer of phospholipids with embedded proteins. It contains cholesterol that determines the membrane's fluidity, permeability and activity of membrane proteins. Changes in membrane cholesterol are implicated in diseases such as Alzheimer's and cancer, demanding its investigation.
NR12S is a fluorophore that exhibits emission maxima at 560 and 630 nm dependent on the liquid order of the membrane. Incorporation of the dye in a liquid ordered phase (increased cholesterol) shifts the emission to 560 nm, whereas in a liquid disordered phase (lower cholesterol) it emits at 630 nm. Hence, the 560/630 ratio correlates with the membrane cholesterol content.
Inhibition of cholesterol synthesis by lovastatin or incubation with methyl-§-cyclodextrin decreased membrane cholesterol in hematological cells and was reported by decreases in the 560/630 ratio. Both emissions were conveniently measured on the filter-based FLUOstar® Omega microplate reader and could be analyzed with one-click using the MARS analysis software.