Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast

Le, X. Chris; Tan, Woei; Scaman, Christine H.; Szpacenko, Adam; Arriaga, Edgar A.; Zhang, Yanni; Dovic̀hi, Norman J.; Hindsgaul, Ole; Palcic, Monica M.

doi:10.1093/glycob/9.3.219

Cited by 32 publications

(42 citation statements)

References 28 publications

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“…Microcapillary methods can be used for either isoelectric focusing of whole cells or the electrophoretic separation of intracellular analytes from a single cell after lysis (15,16,133,139,211). Microcapillary separations are relatively rapid, and reactions can be monitored using either UV absorption or fluorescence detection methods.…”

Section: Microcapillary Electrophoresismentioning

confidence: 99%

“…Fluorescence-based monitoring can be used to observe whole-cell staining with diagnostic fluorescent dyes or to characterize metabolic transformations of fluorescent enzyme substrates in single cell lysates (Fig. 9) (16,139,211).…”

Section: Microcapillary Electrophoresismentioning

confidence: 99%

“…Microcapillary-based methods for performing biochemical separations of the contents of individual cells, including yeast, on May 11, 2018 by guest http://mmbr.asm.org/ have also been described (98,132,133,134,139). Individual cells may be delivered into a capillary tube by suction forces or electroosmotic flow, with monitoring via microscopy (134,139).…”

Section: Microcapillary Electrophoresismentioning

confidence: 99%

“…Individual cells may be delivered into a capillary tube by suction forces or electroosmotic flow, with monitoring via microscopy (134,139). After a cell is loaded into the capillary, it is lysed to release the cell contents for analysis.…”

Section: Microcapillary Electrophoresismentioning

confidence: 99%

“…After a cell is loaded into the capillary, it is lysed to release the cell contents for analysis. The addition of surfactant may be sufficient to lyse mammalian cells, but thick-walled microorganisms such as S. cerevisiae may require spheroplasting first (134,139). Krylov et al (133) found that if electrophoresis was begun immediately following cell lysis, enzymatic activity was effectively "quenched" and artifacts stemming from nonmetabolic enzymatic activity or other degradative processes were suppressed.…”

Section: Microcapillary Electrophoresismentioning

confidence: 99%

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Single-Cell Microbiology: Tools, Technologies, and Applications

Brehm-Stecher

Johnson

2004

Microbiol Mol Biol Rev

452

316

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The field of microbiology has traditionally been concerned with and focused on studies at the population level. Information on how cells respond to their environment, interact with each other, or undergo complex processes such as cellular differentiation or gene expression has been obtained mostly by inference from population-level data. Individual microorganisms, even those in supposedly “clonal” populations, may differ widely from each other in terms of their genetic composition, physiology, biochemistry, or behavior. This genetic and phenotypic heterogeneity has important practical consequences for a number of human interests, including antibiotic or biocide resistance, the productivity and stability of industrial fermentations, the efficacy of food preservatives, and the potential of pathogens to cause disease. New appreciation of the importance of cellular heterogeneity, coupled with recent advances in technology, has driven the development of new tools and techniques for the study of individual microbial cells. Because observations made at the single-cell level are not subject to the “averaging” effects characteristic of bulk-phase, population-level methods, they offer the unique capacity to observe discrete microbiological phenomena unavailable using traditional approaches. As a result, scientists have been able to characterize microorganisms, their activities, and their interactions at unprecedented levels of detail

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Section: Microcapillary Electrophoresismentioning

confidence: 99%

Section: Microcapillary Electrophoresismentioning

confidence: 99%

Section: Microcapillary Electrophoresismentioning

confidence: 99%

Section: Microcapillary Electrophoresismentioning

confidence: 99%

Section: Microcapillary Electrophoresismentioning

confidence: 99%

See 3 more Smart Citations

Single-Cell Microbiology: Tools, Technologies, and Applications

Brehm-Stecher

Johnson

2004

Microbiol Mol Biol Rev

452

316

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Correlating cell cycle with metabolism in single cells: Combination of image and metabolic cytometry

et al. 1999

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Background:We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. Methods: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. Results: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G 1 phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G 2 /M cells. Conclusions: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle. Cytometry 37: 14-20, 1999. 1999

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Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell

Zhang

Cook³

et al. 2001

Electrophoresis

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Capillary Sodium dodlecyl sulfate (SDS)-DALT an (abbreviation for Dalton) electrophoresis was applied to analysis of proteins in single HT29 human colon adenocarcinoma cells. A vacuum pulse was employed to introduce a single cell into the coated capillary. Once the cell was lysed, proteins were denatured with SDS, fluorescantly labeled with 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ), and then separated by using 8% pullulan as the sieving matrix. This method offers a few advantages for single-cell protein analysis. First, it provides reproducible separation of single-cell proteins according to their size. Based on comparison with the migration time of standard proteins, most components from a single HT29 cancer cell have molecular masses within the range of 10-100 kDa. Second, as a one-dimensional separation method, it gives fairly good resolution for proteins. Typically, around 30 protein components of a single HT29 cell were resolved, indicating that this method has similar peak capacity to SDS-polyacrylamide gel electrophoresis (PAGE). Third, this method shows high detection sensitivity and wide dynamic range, which is important because of the wide range of protein expression in living systems. Detection limits for standard proteins ranged from 10(-10) to 10(-11) M. Finally, this method provides much higher speed than classical gel electrophoresis methods, and it provides automated anlysis of cellular proteins at the single-cell level; the separation is complete in 30 min and the entire analysis takes approximately 45 min.

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Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast

Cited by 32 publications

References 28 publications

Single-Cell Microbiology: Tools, Technologies, and Applications

Single-Cell Microbiology: Tools, Technologies, and Applications

Correlating cell cycle with metabolism in single cells: Combination of image and metabolic cytometry

Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell

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