Cytometry

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. 

CLARKE R G, LUND E K, JOHNSON I T aud PINDER A c (2000) Apoptosis cau he detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, hut uot hy TUNEL assay or suh-GO DNA content . Cytometry, 39 141-50. 

Schwille, P., Korkach, J. and Webb, W. W. (1999) Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes. Cytometry, 36, 176-182. 

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). 

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