C6 NBD galactosylceramide, an active derivative of galactosylceramide tagged with fluorescent nitrobenzoxadiazole (NBD), serves as a substrate for neutral β-glycosylceramidase (GCase) in studies of intracellular localization and metabolism of galactosylceramide (Ex=nm, Em=525) [1].

C6-NBD-glucosylceramide (4 μM) undergoes transport to the Golgi apparatus in HT29 cells [1]. According to our recommended protocol, transcytosis of exogenous C6-NBD-GaICer follows these steps after endocytosis: Initially, C6-NBD-glucosylceramide is inserted at 10°C. Subsequently, cells are subjected to three cold HBSS rinses and maintained at 37°C in HBSS to facilitate endocytosis. After 10 minutes, residual probe on the cell surface is eliminated through either two (apical) or three (basal) 20-minute BSA washes at 10°C. Lipid analysis using one set of filters quantifies the endocytosis, while a second set incubated for either 0.5 or 1 hour at 37°C in HBSS + BSA measures the reemergence of intracellular C6-NBD-glucosylceramide on the cell surfaces. Post-incubation, a 10°C BSA wash is conducted before extracting the NBD lipids into chloroform/methanol from both apical and basal media, as well as the cells, which are then analyzed and quantitated by TLC.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.