An improved assay for measuring the transverse redistribution of fluorescent phospholipids in plasma membranes

The internalization of fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phospholipids from the plasma membrane can be assessed by the irreversible quenching of analogues in the outer leaflet by dithionite. Here we have utilized this assay to follow the redistribution of short-chain C6-NBD-sphingomyelin and C6-NBD-phosphatidylserine from the cell membrane of human gingiva fibroblasts. The significant uptake of dithionite across the plasma membrane and the subsequent reduction of NBD-analogues exposed to the cytoplasmic lumen does not allow an accurate measurement of the amount of internalized lipid probes even at low temperature. We could show that a precise determination can be achieved by extraction of analogues remaining in the exoplasmic half by a short pretreatment with bovine serum albumin prior to addition of dithionite. The fluorescence of those analogues localized to the cytoplasmic lumen was slowly destroyed by permeating dithionite. The fluorescence of those NBD-probes which are localized in the inner layer of intracellular vesicles remained almost unaffected in the time course of the assay. Thus, this approach allows to distinguish between different routes of internalization of NBD-phospholipids from the plasma membrane.