Flow cytometry staining

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.

Flow cytometry A method of measuring the number of cells in a sample, the percentage of five cells in a sample, and certain characteristics of cells, such as size, shape, and the presence of tumor markers on the cell surface. The cells are stained with a light-sensitive dye, placed in a fluid, and passed in a stream before a laser or other type of light. The measurements are based on how the light-sensitive dye reacts to the light. 

Ladies, G.S., et al., Phase two of an interlaboratory evaluation of the quantification of rat splenic lymphocyte subtypes using immunofluorescent staining and flow cytometry, Toxicol. Methods, 8, 87, 1998. 

One of the first applications developed for flow cytometry was cell cycle analysis.2 There are numerous intercalating fluorescent DNA and RNA staining reagents that can be used to determine the amount of DNA in cells, an indicator of cell cycle stage and progression, as demonstrated in Figure 7.3. Nucleic acid dyes may be selective for DNA... 

Fig. 10.19 Lack of toxic effects of CM fullerene on breast epithelial cells. Cm does not inhibit cell proliferation. MCF 10A and (A) MDA MB 231 (B) breast cancer cell lines were cultured either in the absence or presence of methanol C60 and cell proliferation was assayed by crystal violet staining. Control, no C 1 Opg Cm, A 50 pg Cm, X 250 pg CM. (C). MDA MB 231 cells were simultaneously stained with calcein and ethidium using a live-dead assay kit. Lack of red-colored cells and the presence of cells stained in green indicate the lack of toxicity (D). MDA MB 231 cells were either untreated (open box) cultured with varying amounts 10 (gray ), 50 (patterned ) and lOOpg (filled ) of C60 for 48 h and analyzed for cell cycle progression by flow cytometry (Levi et al., 2006) (See Color Plates)...

The problem has now been solved, and it is possible to measure the phase angle of the probe as the cells pass through the laser beam.09,40) While these measurements have not yet been applied to Ca2+, the method has been validated with standard beads and stained cells. In our opinion, this new flow cytometry parameter, and our increasing knowledge of lifetimes of probes, will result in the increased use of flow cytometry for studies of intracellular physiology, in addition to the current emphasis in immunology, cell activation, and ploidy. 

Fluorochromes are stains that interact directly with cellular components, or are used to form conjugates with antibodies or ligands, yielding fluorescent reporter molecules. O Table 13-1 lists a selection of fluoro- D Table 13-1 Fluorochromes for flow cytometry ... 

Fixation procedures are necessary when biohazardous samples are analyzed, and are sometimes used to allow access of membrane impermeant fluorochromes, or to stabilize samples for short-term storage. Optimal fixatives are those that have low autofluorescence and do not significantly affect staining. Paraformaldehyde, at concentrations of 0.5-2%, and ethanol (70%, 4 C) are widely used fixatives for flow cytometry. Combinations of paraformaldehyde with Triton X-100 or saponin have been employed in procedures that fix and permeabilize cells. 

The analysis of cell-cycle progression was one of the earliest applications of flow cytometry (for review, see Darzynkiewicz et al., 2004). In this assay, fluorescence signals from cells stained with DNA-binding fluorochromes are plotted as DNA content histograms that may be analyzed by using histogram deconvolution software to quantify cell-cycle phase distributions (Rabinovitch 1994). Fluorochromes that are useful for this purpose are the plasma membrane-impermeant DNA stains, propidium iodide (PI),... 

The following basic protocols may be used alone or combined with other staining procedures in multiparameter flow cytometry experiments. Although they are illustrated with data from cells that proliferate in suspension, these protocols may be easily modified for the analysis of cells isolated from tissues or adherent cells in culture, by incorporating an initial step for the preparation of single cell suspensions. The assays are conducted at room temperature, unless otherwise noted. 

Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139 271-279. 

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so on) can be used to detect or measure any cell constituent, provided that the tag reacts specifically and stoichiometrically with the cellular constituent in question (2). Today, the repertoire of fluorescent probes is expanding almost daily see Chapter 14). One area that has benefited from the ever-increasing number of fluorescent probes is flow cytometry. 

Sasaki, K. and Kurose, A. (1992) Cell staining for flow cytometry. Nippon Rinsho. 50, 2307-2311. 

Deitch, A. D., Law, H., and DeVere White, R. (1992) A stable propidinm iodide staining procednre for flow cytometry. J. Histochem. Cytochem. 30,967-972. Taylor, I. W. (1980) A rapid single step staining techniqne for DNA analysis by flow microflnorimetry. J. Histochem. Cytochem. 28,1021-1024. 

Davis KA, Lin Y, Abrams B et al (1998) Staining of a cell surface human CD4 with 2 F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic Acids Res 26 3915-3924... 

In another experimental setting in vitro data on the cell surface antigen expression were obtained for PBMCs isolated from chronic lymphocytic leukemia (CLL) patient blood samples and cultured in the presence of DIMS (according to standard procedures). Cultured cells were collected and stained with the corresponding antibodies for detection of the expression of cell surface antigens. Subsequendy they were analyzed by flow cytometry. 

Defective phagocytosis by neutrophils in CGD can be demonstrated by the classical nitroblue terazolium test by the absence of dark-blue staining of the cytoplasm, in contrast, normal neutrophils with efficient phagocytic function exhibit dark-blue cytoplasmic staining (84). Flow cytometry is also a powerful tool for the study of CGD (85). 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

Wilson, G D, McNally, N. J, Dunphy, E, Karcher, H., and Pfragner, R (1985) The labelling index of human and mouse tumours assessed by bromo-deoxyuridine staining in vitro and in vivo and flow cytometry Cytometry 6, 641-647. 

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