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.