Metabolic cytometry. Glycosphingolipid metabolism in single cells.
Glycosphingolipids are found on all vertebrate cells and constitute major cell surface determinants on all nerve cells, where they contribute to cellular diversity and function. We report a method for the analysis of glycosphingolipid metabolism in single cells. The ganglioside GM1 was tagged with the fluorescent dye tetramethylrhodamine. This labeled compound was taken up and metabolized by a culture of pituitary tumor (AtT-20) cells. After 50 h, the cells were formalin fixed. Cells were aspirated into a fused-silica capillary and lysed, and components were separated by capillary electrophoresis with a laser-induced fluorescence detector. All metabolic products that retained the fluorescent dye could be detected at the low-zeptomole level. A total of 54 AtT-20 cells were individually analyzed using this procedure. The electrophoretic profiles were remarkably reproducible, which facilitated identification of components based on the migration time of fluorescently labeled standards. Eleven components were detected, and the average peak height of these components spanned more than 2 orders of magnitude, so that trace metabolites can be detected in the presence of abundant components. The most highly abundant components generated 10% relative standard deviation in normalized abundance. The average cell took up roughly 2 amol (10(6) copies) of the labeled substrate. This method allows determination of cell-to-cell diversity and regulation of glycosphingolipid metabolism.
Whitmore
Colin D
CD
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, USA.
Hindsgaul
Ole
O
Palcic
Monica M
MM
Schnaar
Ronald L
RL
Dovichi
Norman J
NJ
D000818:Animals; D045744:Cell Line, Tumor; D002477:Cells / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006028:Glycosphingolipids / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D012235:Rhodamines / Q000032:analysis
D005456:Fluorescent Dyes; D006028:Glycosphingolipids; D012235:Rhodamines; C005358:tetramethylrhodamine