Rapid mix flow cytometry

Summary of Experimental Results of LRG Dissociation Kinetics Measured by Small Volume Rapid Mix Flow Cytometry... 

Wu, Y., Buranda, T., Simons, P. C., Lopez, G. P., Mclntire, W. E., Garrison, J. C., Prossnitz, E. R., and Sklar, L. A. (2007a). Rapid mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides. Anal. Biochem. In 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. 

In summary, flow cytometry is clearly useful in evaluating macrophages and their role in toxicity. A major advantage of this technology is the rapid and accurate identification of subpopulations of responding cells from within a mixed population. There is no doubt that the utility of flow cytometry will increase in the future as new fluorescence probes are developed that allow investigators to more clearly assess various macrophage characteristics and the response of these cells to xenobiotics. 

Fig. 11.12. The use of gel microdroplets and flow cytometry to assay drug sensitivity of bacterial cells. The figure shows side scatter and green fluorescence contour plots of gel microdroplets (GMDs) containing E. coli cells that have been stained with fluorescein isothiocyanate for total protein. The microdroplets have been analyzed in the flow cytometer either at time 0 or 2 h after incubation in control medium (left plots) or medium containing penicillin (right plots). A model system was created by mixing two strains of bacteria (susceptible or resistant to penicillin). The data show that a small subpopulation of resistant cells could be detected within 2 h because of its rapid growth in comparison to susceptible cells. From Weaver et al. (1991).

An additional indirect assay of neutrophil motility is the actin polymerization assay. Chemoattractants induce a rapid, transient actin polymerization response in neutrophils (reviewed in [289, 391]), and since actin polymerization is required for migration to occur, it is often used as an indication of chemotactic capability. Several approaches have been developed to measure this response (reviewed in [289, 391]) (Table 2). Most commonly, cells in suspension are mixed with a putative chemoattractant then fixed and stained with a fluorescent phaUotoxin that binds to polymerized actin, but not monomeric actin. The bound fluorescence is quantified using flow cytometry or spectrofluorometry. The actin polymerization response to many neutrophil chemoattractants is rapid, reaching a maximum within 10 s of stimulus addition at 37° C. As with the cell polarization assay, no information about the migratory properties of the cells is obtained with the actin polymerization assay, only the likelihood of chemotactic activity is assessed. Like the cell polarization assay, the actin polymerization assay is useful for initial screening of chemoattractants, but must be followed up with an actual measurement of motility. 

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