C6 NBD Galactosylceramide (d18:1/6:0)

C6 NBD galactosylceramide is an active derivative of galactosylceramide that is tagged with fluorescent C6 nitrobenzoxadiazole (C6 NBD). C6 NBD galactosylceramide can be used as a substrate for neutral β-glycosylceramidase (GCase) to study intracellular localization and metabolism of galactosylceramide (Ex=nm, Em=525)[1].

Research Areas >> Others

Signaling Pathways >> Others >> Others

C6-NBD-glucosylceramide (4 μM) is transported to the Golgi apparatus in HT29 cells[1]. Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)[2]. Transcytosis of Exogenous C6-NBD-GaICer after Endocytosis: 1. C6-NBD-glucosylceramide is inserted at 10°C. 2. Cells are rinsed three times with cold HBSS' and incubated at 37°C in HBSS' to allow endocytosis. 3. After 10 min, the probe remaining on the cell surface is removed by two (apical) or three (basal) BSA washes for 20 min at 10°C. 4. One set of filters is used for lipid analysis to quantitate endocytosis. A second set of filters is further incubated for 0.5 or 1 h at 37°C in HBSS' + BSA, to assay for reappearance of intracellular C6-NBD-glucosylceramide on either cell surface. 5. The incubations are followed by a 10°C BSA wash, after which the NBD lipids from the combined apical media, basal media, and the cells were extracted into chloroform/methanol, analyzed by TLC, and quantitated.

[1]. J W Kok, et al. Sorting of sphingolipids in the endocytic pathway of HT29 cells. J Cell Biol. 1991 Jul;114(2):231-9.

[2]. I van Genderen, et al. Differential targeting of glucosylceramide and galactosylceramide analogues after synthesis but not during transcytosis in Madin-Darby canine kidney cells. J Cell Biol. 1995 Nov;131(3):645-54.