Metabolic cytometry: Capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons

David C. Essaka, Jillian Prendergast, Richard B. Keithley, Monica M. Palcic, Ole Hindsgaul, Ronald Lee Schnaar, Norman J. Dovichi

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)2799-2804
Number of pages6
JournalAnalytical Chemistry
Volume84
Issue number6
DOIs
StatePublished - Mar 20 2012

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Capillary electrophoresis
Glycosphingolipids
Neurons
Fluorescence
Color
Glycolipids
Crosstalk
Rats
Detectors
Lasers
Substrates
Experiments
Metabolic Networks and Pathways
tetramethylrhodamine
BODIPY-LacCer
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Metabolic cytometry : Capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons. / Essaka, David C.; Prendergast, Jillian; Keithley, Richard B.; Palcic, Monica M.; Hindsgaul, Ole; Schnaar, Ronald Lee; Dovichi, Norman J.

In: Analytical Chemistry, Vol. 84, No. 6, 20.03.2012, p. 2799-2804.

Research output: Contribution to journalArticle

Essaka, David C. ; Prendergast, Jillian ; Keithley, Richard B. ; Palcic, Monica M. ; Hindsgaul, Ole ; Schnaar, Ronald Lee ; Dovichi, Norman J. / Metabolic cytometry : Capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons. In: Analytical Chemistry. 2012 ; Vol. 84, No. 6. pp. 2799-2804.
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