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ABSTRACT: A capillary electrophoresis system with ultrasensitive two-color laser-induced fluorescence detection was used to probe the effect of ionic strength on single cell separations of glycosphingolipids. Differentiated PC12 cells were incubated with two ganglioside substrates tagged with different fluorophores within the BODIPY family such that two distinct metabolic patterns could be simultaneously monitored. Aspiration of single differentiated PC12 cells suspended in a phosphate-buffered saline solution showed excessive peak dispersion, poor resolution, and peak efficiencies below 100,000 theoretical plates. Aspiration of single differentiated PC12 cells suspended in deionized water corrected peak dispersion. Average peak efficiencies ranged between 400,000 and 600,000 theoretical plates. Improved performance was due to the dilution of the high salt concentrations inside of single neuronal-like cells to produce field amplified sample stacking. Single cell separations showed the highest resolution when aspiration of single differentiated PC12 cells suspended in deionized water were separated using a running buffer of high ionic strength. The improvement in resolution allowed for the identification of analytes not previously detected in single cell metabolism studies.
Talanta 07/2013; 111C:206-14. · 3.79 Impact Factor
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ABSTRACT: A capillary electrophoresis system with an ultrasensitive three-color laser-induced fluorescence detector was constructed for the simultaneous measurement of glycosphingolipids conjugated with a family of BODIPY fluorophores. The compounds were separated by capillary electrophoresis and detected by laser-induced fluorescence excited within a sheath-flow cuvette. Diode-pumped solid-state lasers operating at 473 nm and 532 nm, and a diode laser operating at 633 nm were used to excite glycosphingolipids tagged with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 fluorophores. Detection limits were 34 ± 1 molecules, 67 ± 7 molecules, and 36 ± 13 molecules of BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled glycosphingolipids. Separation efficiencies were typically one million theoretical plates. To test the ability of the system to analyze cellular contents in an in vitro biological model, differentiated PC12 cells were co-incubated with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled lactosylceramide substrates. Cells were homogenized. The metabolic products originating from the glycosphingolipid substrates were simultaneously analyzed using the system.
The Analyst 11/2012; · 4.23 Impact Factor
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ABSTRACT: Several glycosphingolipids were labeled with the fluorphore Bodipy-Fl and analyzed using capillary electrophoresis with laser-induced fluorescence detection. GM1-, LacCer-, and Cer-Bodipy-Fl were prepared through acylation using the N-hydroxysuccinimide ester of Bodipy-Fl. Several other glycosphingolipids including GT1a-, GD1a-, GM2-, GM3-, GD3-, and GlcCer-Bodipy-Fl were enzymatically synthesized. Micellar electrokinetic capillary chromatography with a TRIS/CHES/SDS/α-cyclodextrin buffer produced better separation than an established borate/deoxycholate/methyl-β-cyclodextrin buffer. The nine Bodipy-Fl-labeled glycosphingolipid standards were separated in under 5 min, theoretical plate counts were between 640,000 and 740,000, and the limit of detection was approximately 3 pM or 240 ymol analyte injected onto the capillary.
Journal of chromatography. A 03/2012; 1229:268-73. · 4.19 Impact Factor
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ABSTRACT: Cell-to-cell heterogeneity in ganglioside catabolism was determined by profiling fluorescent tetramethylrhodamine-labeled GM1 (TMR-GM1) breakdown in individual primary neurons and glia from the rat cerebellum. Cells isolated from 5 to 6 day old rat cerebella were cultured for 7 days, and then incubated for 14 h with TMR-GM1. Intact cells were recovered from cultures by mild proteolysis, paraformaldehyde fixed, and subjected to single cell analysis. Individual cells were captured in a capillary, lysed, and the released single-cell contents analyzed by capillary electrophoresis with quantitative laser-induced fluorescent detection of metabolites. Non-neuronal cells on average took up much more exogenous TMR-GM1 than neuronal cells, and catabolized it more extensively. After 14 h of incubation, non-neuronal cells retained only 14% of the TMR products as GM1 and GM2, compared to >50% for neurons. On average, non-neuronal cells contained 74% of TMR-labeled product as TMR-ceramide, compared to only 42% for neurons. Non-neuronal cells retained seven times as much TMR-GM3 (7%) compared to neuronal cells (1%). To confirm the observed single cell metabolomics, we lysed and compared TMR-GM1 catabolic profiles from mixed neuron/glial cell cultures and from cultures depleted of non-neuronal cells by treatment with the antimitotic agent cytosine arabinoside. The lysed culture catabolic profiles were consistent with the average profiles of single neurons and glia. We conclude that the ultrasensitive analytic methods described accurately reflect single cell ganglioside catabolism in different cell populations from the brain.
Neurochemical Research 03/2012; 37(6):1308-14. · 2.24 Impact Factor
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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.
Analytical Chemistry 03/2012; 84(6):2799-804. · 5.86 Impact Factor
