Flow cytometric analysis

Borzi RM, Mazzetti I, Macor S, et al. Flow cytometric analysis of intracellular chemokines in chondrocytes in vivo constitutive expression and enhancement in osteoarthritis and rheumatoid arthritis. FEBS Lett 1999 455(3) 238-242. 

Fuchs, B. M. Wallner, G. Beisker, W. Schwippl, I. Ludwig, W. Amann, R. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 1998, 64, 4973 1982. 

Reader, S., H.B. Steen, and F. Denizeau. 1994. Intracellular calcium and pH alterations induced by tri-n-butyltin chloride in isolated rainbow trout hepatocytes a flow cytometric analysis. Arch. Biochem. Biophys. 312 407-113. 

Pestka, J. J., Yan D., and King L.E. Flow cytometric analysis of the effects of in vitro exposure to vomitoxin (deoxynivalenol) on apoptosis in murine T, B and IgA+ cells. Food Chem. Toxicol. 32, 1125, 1994. 

Koyama, S., Maruyama, T., and Adachi, S. (1999). Expression of epidermal growth factor receptor and CD44 splicing variant sharing exons 6 and 9 on gastric and esophageal carcinomas a two flow-cytometric analysis. J. Cancer Res. Clin. Oncol. 125, 47—54. 

J. T. Ransom, D. L. DiGiusto, and J. Cambier, Flow cytometric analysis of intracellular calcium mobilization, Methods Enzymol. 141, 53-63(1987). 

G. L. Rossi, D. J. Young, S. I. Wasserman, and K. E. Barrett, Calcium mobilization in activated mast cells monitored by flow cytometric analysis. Agents Actions 31, 257-262 (1990). 

B. Goller and M. Kubbies, UV Lasers for flow cytometric analysis HeCd versus argon laser excitation, J. Histochem. and Cytochem. 40(4), 451 —456 (1992). 

In addition to the detection methods described hitherto, binding and uptake of targeted drug delivery systems can be determined by flow cytometric analysis. Similar to conventional fluorimetric techniques, flow cytometry... 

Mattiasson G, Friberg H, Hansson M, Elmer E, Wieloch T. 2003. Flow cytometric analysis of mitochondria from CAl and CA3 regions of rat hippocampus reveals differences in permeability transition pore activation. J Neurochem 87 532-544. 

Ratinaud MH, Leprat P, Julien R. 1988. In situ flow cytometric analysis of nonyl acridine orange-stained mitochondria from splenocytes. Cytometry 9 206-212. 

Keep in mind that exocytosis might occur during sample preparation (e.g., preparing cells for flow cytometric analysis). After the experiment (and prior to analysis) cells should be kept below 4°C to block all active processes (such as exocytosis). Exocytosis might also be blocked with NEM (see section Caveolae-Mediated Endocytosis and Caveolae-Like Endocytosis Pharmacological Inhibitors ). 

Disadvantages Associated with Flow Cytometric Analysis... 

Successful flow cytometric analysis depends on adequate sample preparation (see Chapters 30-31), appropriate selection of probes or markers (see Subheading 2.), instrumentation, and data display and analysis. Each of these areas is interrelated and requires adequate attention to avoid the introduction of artifacts and misinterpretation of results. Flow cytometers tend to be excessively complicated and require a skilled operator for alignment and calibration, though manufacturers are introducing more compact, user-friendly data acquisition and image processing systems. 

Fluorescence staining for flow cytometric analysis falls into three categories methods in which a fluorescent ligand accumulates on or within the cell (see Chapters 36,38) methods that require the ligand to interact with a cellular component to release the fluorophore or result in light emission (see Chapters 34,39) and methods that rely on fluorophore-coupled antibody binding (see Chapters 32,33). 

Coon, J. S., Landay, A. L., and Weinstein, R. S. (1986) Flow cytometric analysis of paraffin-embedded tumors implications for diagnostic pathology. Human Pathol. 17, 435 37. 

A variety of assays have been developed to quantify phagocytic activity. These include direct microscopic visualization (2,3), spectrophotometric evaluation of phagocytized paraffin droplets containing dye (4), scintillation counting of radiolabeled bacteria (5), fluorometric (6), and flow cytometric analysis of fluorescent particles (7-13). The flow cytometric assay offers the advantage of rapid analysis of thousands of cells and quantification of the internalized particle density for each analyzed cell. The assay may be performed with purified leukocyte preparations (7-13) or anficoagulated whole blood (14,15). 

A major limitation of flow cytometric analysis is that it provides data from individual cells at a single point in time and the same cells are not available for further analysis once they have passed through the flow cell of the instrument. Therefore, it is not possible to monitor a given cell over time for changes in fluorescence intensity or distribution of fluorescence signal. Such studies require microinjection of the fluorochrome into individual cells and fluorescence microscopy analysis. 

Finney, D. A. and Sklar, L. A. (1983) Ligand/receptor internalization a kinetic, flow cytometric analysis of the internalization of A-formyl peptides by human neutrophils. Cytometry 4, 54-60. 

As stated earlier, activation of endothelial cells by pro-inflammatory stimuli leads to the expression of cell adhesion molecules and cytokines such as IL-6 and IL-8. The expression and hence modulation of surface expressed adhesion molecules by e.g. targeted delivery of inhibitors of NFkB, can be measured using flow cytometric analysis or whole cell ELISA techniques. Cytokine production can be measured in the supernatant of cultured cells or in biological fluids. Furthermore, competitive or quantitative RT-PCR analysis of mRNA levels of cell adhesion molecules or cytokines, allows the transcriptional activity of the genes of interest to be estimated. 

Figure 6.2 Cell cycle distributions of MCF-7 cells after treatment with TSA for 24 hours were determined by performing flow cytometric analysis. Following treatment of cells with 50 nM and 1 P-M of TSA, the samples were analyzed on a Becton-Dickinson FACStarP "" instrument and the percentage of nuclei with C], S, and C2/M DNA content was determined. p21 protein was also correspondingly increased at various intervals.

Evenson DP, Janca FC, Jost LK, et al. 1989b. Flow cytometric analysis of effects of 1,3,-dinitrobenzene on rat spermatogenesis. J Toxicol Environ Health 28 81-98. 

Ginsberg, M. H., Frelinger, A. L., Lam, S. C. T., Forsyth, J., McMillan, R., Plow, E. F., and Shattil, S. J., Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein llb-llla with conformation specific monoclonal antibodies. Blood 76, 2017—2023... 

Vowells, S. J., Sekhsaria, S., Malech, H. L., Shalit, M., and Fleischer, T. A., Flow cytometric analysis of the granulocyte respiratory burst A comparison study of fluorescent probes. J. Immunol Methods 178, 89-97 (1995). 

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