Metabolic cytometry

Krylov, S. N., Arriaga, E. A., Chan, N. W., Dovichi, N. J. and Palcic, M. M. Metabolic cytometry monitoring oligosaccharide biosynthesis in single cells by capillary electrophoresis. Anal Biochem, 283,133, 2000. 

This research group coined the terms chemical cytometry and metabolic cytometry in 1999. Metabolic cytometry is a form of chemical cytometry that monitors biosynthetic and biodegradation enzymatic cascades in a single cell. 

In metabolic cytometry, cells are incubated with a substrate that is tagged with a highly fluorescent dye we prefer tetramethylrhodamine because of its excellent spectroscopic properties and its compatibility with the frequency-doubled neodymium YAG laser. This substrate is prepared at high concentration and undergoes chromatographic purification to eliminate fluorescent impurities. 

FIGURE 21.13 Metabolic cytometry. A substrate (top) is fluorescently labeled (star). This substrate can undergo biosynthesis (left) or biodegradation (right). As long as the fluorescent tag remains intact, the metabohc products can be monitored by chemical cytometry. 

We have also synthesized the biodegradation products for this compound, including the asialo Gai. and Ga2, which are used as standards to tentatively identify metabolic cytometry products based on comigration. 

FIGURE 21.16 Metabolic cytometry analysis of three cells. AtT20 cells were incubated with the substrate of Figure 21.15, aspirated into a capillary, lysed, and analyzed by CE. 

Figure 21.16 presents metabolic cytometry data generated from single AtT20 cells. In these experiments, a cell is incubated with the substrate. The substrate is taken up, which can be confirmed by fluorescence microscopy. Enzymatic transformations convert the substrate to products. To analyze the products, the cell is aspirated into a capillary, lysed, components are separated by CE, and products... 

Essaka, D. C. Prendergast, J. Keithley, R. B. Palcic, M. M. Hindsgaul, O. Schnaar, R. L. Dovichi, N. J. Metabolic cytometry Capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons. Anal. Chem. 2012, 84, 2799-2804. 

Recently, Beatty and Tirrell [201] relied on the simultaneous or sequential addition of two reactive Met analogs, Aha and Hpg, to enable the fluorescent tagging of two protein populations within cells. The first demonstration of two-dye labeling of metabolically tagged cells was described in 2007 by Chang and co-workers [202], who used flow cytometry to show that cells treated with two reactive sugars could be labeled with distinct fluorophores. 

A key feature of apoptosis is that, similar to the cell cycle, the duration of apoptosis is variable and the process is asynchronous in most cell populations. Consequently, there are variable proportions of cells in distinct phases of apoptosis and a sample, collected from a culture in which apoptosis has been induced, will contain cells in all the phases of apoptosis. This could represent a major technical problem in the investigation of apoptosis and requires measurement of metabolic apoptotic changes concomitantly with established markers of apoptosis progression. Using the same cellular model described above, we investigated metabolic alterations induced by X-rays using flow cytometry and a panel of fluorochromes... 

Cell cytometry is a technique to determine the populations of cells in particular parts of their metabolic cycles. One places the cells in a medium containing nutrient molecules that have been tagged with fluorescent dyes. The nutrient is then washed out and fluid containing the cells is passed through a capillary, where UV light is used to make the dye molecules within the cells fluoresce. The capillary has a valve downstream that switches to allow the fluorescing cells to be collected in another flask to concentrate them. 

There has also been an increase in the ability to measure the intracellular pH, by flow cytometry with pH-dependent fluorescence indicators (Oz-kan and Mutharasan, 2002) and nuclear magnetic resonance. There are several reports that relate the intracellular pH value to cell behavior, including metabolism, apoptosis, and specific growth rate (Cherlet and Marc, 1998). Such offline measurements help to interpret the physiological state of the cells, but still cannot be used as control systems. 

Owing to its applications in homeland security, sterilization validation, and astrobiology, bacterial spore detection has become a hot field. However, direct detection of bacterial spores can be challenging for the same reasons that make endospores difficult to irradicate. The tough spore coat is impermeable to staining techniques, so most microscopy and flow cytometry methods are not useful. Endospores are also highly resistant to lysis, meaning that common DNA extraction protocols are difficult to perform. The lack of measurable metabolism renders microcalorimetry and cellular respiration techniques ineffective. 

Flow cytometry approach to study neuronal suspensions [31] allows to measure directly an increase in ROS level when the cells are activated by glutamate or its agonists [25,31,32], Activation of glutamate receptors of several kinds was found to result in activation of different metabolic processes. As it is seen from Table 3, ROS signal is suppressed to different extent by different metabolic inhibitors depending on which ligand stimulates the neurons. 

Sampathkumar, S.-G., Jones, M. B., Yarema, K. J. (2006). Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry. Nat. Protocols, 1, 1840-1851. 

The use of this reaction in the biological context was first demonstrated for the chemospecific labeling of Jurkat cell surfaces [63]. Metabolic engineering with N-acetylmannosamine derivative 40c was used to incorporate azides into sialic acid groups on cell surfaces. The cells were then incubated with biotinylated phosphine 49, and the extent of the reaction was quantified by flow cytometry after treatment with fluorescent avidin. Importantly, neither the azide nor the phosphine displayed any reactivity with the cell-surface groups in the absence of its reactive partner. In addition, the cells showed unchanged growth rates after modification. 

DNA flow cytometry is a very useful tool that permits rapid, objective assessment of a large number of cells but may not be readily available. Comet assay, when combined with centrifugal elutriation, can provide a useful in vitro model to study differences in metabolism and the susceptibility of different te,sticular cell types to DNA-damaging compounds. Thus, new findings using these systems should lead to greater knowledge about why a chemical or class of chemicals can cause testicular toxicity. 

The properties of microbial populations result from the characteristics of metabolism and Its control at the level of the Individual cell. Useful descriptions of population behavior may therefore be constructed based on understanding of single-cell metabolism, and, conversely, studies of population properties may be employed to extract Information about single-cell operation. Population balance equations provide the primary mathematical tool for these purposes, and flow cytometry allows experimental access to the distribution of cell states In the population. Application of these methods to bacteria and yeasts Is Illustrated using three exaaq>le systems. 

Arkhipov, S.N. et al.. Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells. Cytometry A, 63, 41, 2005. 

Chemical Cytometry of Proteins, Biogenic Amines, and Metabolic... 

Krylov, S.N., Zhang, Z., Chan, N.W.C., Arriaga, E., Palcic, M.M. and Dovichi, N.J. Correlating cell cycle with metabolism in single cells The combination of image and metabohc cytometry. Cytometry, 1999 37 15-20. 

---