Cytometry, flow

Flow cytometry is a very versatile technique [223] which allows the analysis of more than 104 cells per second [369,370]. This high number results in statistically significant data and distributions of cell properties. Therefore, flow cytometry is a key technique to segregate biomass (into distinct cell classes) and to study microbial populations and their dynamics, specifically the cell cycle [76, 87, 116, 200, 214, 221, 295, 329, 330, 409, 418]. Individual cells are aligned by means of controlled hydrodynamic flow patterns and pass the measuring cell one by one. One or more light sources, typically laser(s), are focused onto the stream of cells and a detection unit(s) measure(s) the scattered and/or fluorescent light (Fig. 24). Properties of whole cells such as size and shape can be 

Among the items that have been measured are vitality, intracellular pH, DNA and RNA content, and specific plasmids [77,408]. Besides nucleic acids [204], other intracellular components can also be analyzed, e.g. storage materials [2, 82,294], enzymes and protein content [6,338], or the cell size [60,61]. The physiological state can also be rapidly assessed [331]. Furthermore, this technique allows the separation of certain cells using a cell sorter, e.g. for strain improvement [28]. The flow cytometry technique has also been used in connection with molecular probes for identification and viability determination of microbial communities [98]. This application of viability estimation is becoming increasingly important [63, 136, 188, 454]. Unfortunately, the equipment is expensive and most of the measurements are tricky and laborious and not well designed for on-line application. 

Jayat and Ratinaud [190] discuss the advantages of multi-parametric analyses taking DNA and other cellular components simultaneously into account see also, e.g. [73, 261]. Flow cytometry is also useful for identification of (new) species [78] or for biomass estimation [299,349]. 

Hewitt et al. [167] have exploited flow cytometry to quantify the impact of fluid mechanical stress on bacterial cultures. A modified technique, called the slit-scan method, allows the determination of cell shapes and of intracellular location of stained components [34]. Image cytometry and fluorescence microscopy are variants for determination of the volume growth of cells or morphology changes and seem to have become increasingly important [60,382,461 ]. 

Flow Cytometry, Wiley-Llss, New York, 202 pp. 

Shapiro, H. M., 1988, Practical Flow Cytometry, 2nd ed., Alan R. Liss, New York, 3S3 pp. 

Flow cytometry provides a method for measuring the emission from single cells as they pass through a laser beam. Flow cytometry can be used with DNA stains to measure the DNA content of cells, with antibodies to detect cell-surface antigens, or with sensing probes to measure intracellular concentrations of ions. 

Hurtubise, R. J., 1990, Phosphorimetry Theory, Instrumentation, and Applications, VCH Publish New York, 370 pp. 

This type of staining can be analysed on any of the modem flow cytometers with the proviso that the machine is equipped with a pulse processing facihty to enable the discrimination of cell doublets. When using the Becton Dickinson FACScan, the propidium iodide should be collected in FL3 rather than FL2 to overcome any crossover with the FTTC in FLl. Routinely the FL3 detector is set to linear amplification should be set at around 400, whilst FLl can be collected with log or linear amplification, and the PMT voltage is usually around 500 (linear) or 300 (log). Controls, either without BrdUrd or the monoclonal antibody, should be included wherever possible to determine the lower limit of detection of the DNA precursor in the bivariate profile. 

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EIA was originally developed as a histological technique to localize specific ceUular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Eluorophore-labeled antibodies have also been used widely for flow cytometry appHcations using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

Flow cytometer cell counts are much more precise and more accurate than hemocytometer counts. Hemocytometer cell counts are subject both to distributional (13) and sampling (14—16) errors. The distribution of cells across the surface of a hemocytometer is sensitive to the technique used to charge the hemocytometer, and nonuniform cell distribution causes counting errors. In contrast, flow cytometer counts are free of distributional errors. Statistically, count precision improves as the square root of the number of cells counted increases. Flow cytometer counts usually involve 100 times as many cells per sample as hemocytometer counts. Therefore, flow cytometry sampling imprecision is one-tenth that of hemocytometry. 

We will first summarize the fluorescence and spectroscopic assays that have been developed for the fluorometer and then describe their applications using flow cytometry. We will summarize research which exemplifies the utility of simultaneous measurement of responses and shows how these methods have provided Information about the signal transduction pathways and activation in neutrophils. 

Figure 5B. Correlation of right-angle light scatter measured by fluorometry and flow cytometry. The top panel shows flow-cytometric data of side scatter of fixed, stained cells during the time course of stimulation by 1-nM (solid line, solid circles) or 0.01-nH (dashed line, open circle) FLPEP. The bottom panel shows the corresponding right-angle light-scatter data acquired pseudo-simultaneously on live cells in the fluorometer. The flow-cytometric data have been averaged, but the fluorometry data are plotted for both duplicates from one donor. Reproduced with permission from Ref. 27. Copyright 1985 Rockefeller University Press.

There is a single assumption in these measurements--namely that the antibody only quenches free ligand. This has been demonstrated specifically by flow cytometry in experiments which show that there is no quenching of ligand on the cell (3). The kinetic analysis depends on rapid interaction of ligand and antibody, which in these experiments is essentially within the mixing time. 

Advantages. The advantages of flow cytometry are numerous. It is the most sensitive of the techniques, able to detect SI,000 fluoro-phores per cell (0.01 nif FLPEP). There is in addition no required total receptor concentration. It is effective for small numbers of... 

Schwartz, A. Quantitative Fluorescein Microbead Standards Flow Cytometry Standards Corporation Research Triangle Park, NC 27709, [1985] technical brochure. 

Whittier JB, McBee K. 1999. Use of flow cytometry to detect genetic damage in mallards dosed with mutagens. Environ Toxicol Chem 18(7) 1557-1563. 

Fig. 3. Flow cytometry analysis. CellQuest software. The fluorescence of 50,000 cells is measured.

Gonzalez-Munoz M, Luque R, Nauwelaers F, Moneo I Detection of Anisakis simplex-induced basophil activation by flow cytometry. Cytometry B Clin Cytom 2006 68 31-36. 

Solanum aviculare 250-ml shake flasks 100 - 600 rpm (3.5 cm throw) t 10 d cellular DNA distribution (flow cytometry) [110]... 

In addition to these time-honoured methods, newer techniques involving bio-luminescense, fluorescent dyes (epifluorescence) and physical methods such as impedance, calorimetry and flow cytometry have been developed. A feature being sought in these methods is rapidity see section 5.6. 

Shapiro H.M. (1990) Flow cytometry in laboratory microbiology new directions. Am Soc Microbiol News, 56, 584-586. 

J.R.M. Smits, L.W. Breedveld, M.W.J. Derksen, G. Kateman, H.W. Balfoort, J. Snoek and J.W. Hofstraat, Pattern classification with artificial neural networks classification of Algae, based upon flow cytometry data. Anal. Chim. Acta, 258 (1992) 11-25. 

J. P. Diaper and C. Edwards. Survival of Staphylococcus aureus in lake water monitored by flow cytometry. Microbiology 140 35 (1994). 

J. Porter, C. Edwards, J. A. W. Morgan, and R. W. Pickup, Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting, Appl. Environ. Microbiol. 59 3327 (1993). 

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