title: Metabolic cytometry: capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons.
abstract: Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: David C
authors.initials: DC
authors.lastname: Essaka
authors.forename: Jillian
authors.initials: J
authors.lastname: Prendergast
authors.forename: Richard B
authors.initials: RB
authors.lastname: Keithley
authors.forename: Monica M
authors.initials: MM
authors.lastname: Palcic
authors.forename: Ole
authors.initials: O
authors.lastname: Hindsgaul
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Schnaar
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D000818:Animals; D001896:Boron Compounds / Q000032:analysis / Q000378:metabolism; D001921:Brain / Q000166:cytology; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D006028:Glycosphingolipids / Q000032:analysis / Q000378:metabolism*; D007790:Lactosylceramides / Q000032:analysis / Q000378:metabolism; D057230:Limit of Detection; D053858:Metabolic Networks and Pathways; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D012235:Rhodamines / Q000032:analysis / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; C527196:BODIPY-LacCer; D001896:Boron Compounds; D005456:Fluorescent Dyes; D006028:Glycosphingolipids; D007790:Lactosylceramides; D012235:Rhodamines; C005358:tetramethylrhodamine