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Background: We set out to develop an assay for the simultaneous analysis of mitochondrial membrane potential and mass using the probes 10-nonyl acridine orange (NAO), MitoFluor Green (MFG), and MitoTracker Green (MTG) in HL60 cells. However, in experiments in which NAO and MFG were combined with orange emitting mitochondrial membrane potential (⌬⌿ m ) probes, we found clear responses to ⌬⌿ m altering drugs for both probes. Methods: The three probes were titrated to determine whether saturation played a role in the response to drugs. The effects of a variety of ⌬⌿ m altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 M and 5 M, using rhodamine 123 at 0.1 M as a reference probe. Results: Incubation of GM130, HL60, and U937 cells with 2,3-butanedione monoxime (BDM), nigericin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitro-phenol (DNP), gramicidin, ouabain, and valinomycin resulted in increases of the fluorescence intensity for MFG or MTG with only a few exceptions. The fluorescence intensity of cells stained with 0.1 M NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 M NAO were similar. Conclusions: MFG, MTG, and NAO appeared poor choices for the membrane potential independent analysis of mitochondrial membrane mass. Considering the molecular structure of these probes that favor accumulation in the mitochondrial membrane because of a positive charge, our results are not surprising. Cytometry 39:203-210, 2000.

The phagocytosis of uniform fluorescent latex particles by pulmonary macrophages in the rat was analyzed by flow cytometric methods. The percentage of phagocytic macrophages and the number of particles per cell were determined from cell-size and fluorescence histograms. A comparison of in vivo and in vitro phagocytosis data showed that the percentage of phagocytic lavaged macrophages reflected the availability of instilled particles. With sodium azide used to model phagocytosis inhibition, it was shown that the percentage of phagocytic cells and the number of particles per cell can be determined simultaneously.

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes in the cellular content of RNA and protein as well as in the RNA-protein, RNA-DNA, and protein-DNA ratios in relation to cell cycle position for large cell populations. An analysis of cycling cell populations (exponentially growing CHO cultures) and noncycling CHO cells arrested in the G1 phase by growth in isoleucine-free medium demonstrated the potential of the technique.

ios Alamos National laboratory, 10s Alamos, New Mexico 87545 (H.C., 1.S.)Using flow cytometry, populations of Chinese hamster ovary cells, asynchronous and synchronized in the cycle, were measured with respect to cellular RNA-and protein-content, as well as cell light scatter properties. Heterogeneities of cell populations were expressed as coefficients of variation (c.v.) in percent of the respective mean values. Populations of cells immediately after mitosis have about 15% higher C.V. than mitotic cell populations, regardless of whether RNA, proteins, or light scatter are measured. These data indicate that cytoplasmic constituents are unequally distributed into the daughter cells during cytokinesis and that unequal cytokinesis generates intercellular metabolic variability during the cycle. An additional increase in heterogeneity, although of smaller degree, occurs during C, phase. Populations of S-phase cells measured in the selective window equivalent to 15-60 min progression through the cycle, i.e., comparable with the mitotic and postmitotic populations, are the most uniform, having 20-30% lower C.V. than the postmitotic cells. Cell progression through S does not involve any significant increase in intercellular variability with respect to RNA or protein content. In unperturbed exponentially growing cultures a critical RNA content is required for G, cells prior to their entrance into S. Thus, the cells equalize in G, with respect to RNA and protein and, during the transition from the period (compartment) of equalization (G,J to the prereplicative compartment (C,@), they exhibit minimal heterogeneity. The cell residence times in the equalization compartments are exponentially distributed, which may reflect the randomness generated by the uneven division of metabolic constituents to daughter cells during cytokinesis. The cell heterogeneities were presently estimated at two metabolic levels, transcription (RNA content) and translation (proteins). The most uniform were populations stained for RNA and the highest variability was observed after staining of proteins. This suggests that the regulatory mechanisms equalizing cells in the cell cycle may operate primarily at the level of DNA transcription.Cell populations of a given type or line are heterogeneous with respect to the cell size as well as protein or RNA content of individual cells. There is also high intercellular variation in rates of progression through the cell cycle (see reviews: Baserga, 1976;Prescott, 1976). Despite this heterogeneity, the mean values of the cell size or content of cell constituents remain constant for a given cell type, from generation to generation. The degree of heterogeneity of the populations also remains constant over generations. Thus, mechanisms do operate during the cell cycle, "equalizing" the populations, i.e., precluding the appearance of cells with extremely low or high protein or RNA-content, or cells traversing the cycle at extreme rates. In addition, there is a control over the mean values of these parameters, so tha...

Fluorescent antibodies are often used to measure the number of receptor sites on cells. The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies. Quenching may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another result in self‐quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity of antibodies relative to one another on a highly labeled cell surface. Since self‐quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase‐sensitive detection method to investigate the impact of self‐quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti‐mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self‐quenching effects expected at high densities of FITC molecules. (This article is a U.S. Government work and, as such, is in the public domain n the United States of America.) © 1996 Wiley‐Liss, Inc.

A new flow-system instrument for quantitative analysis and sorting of microscopic particles, particularly biological cells, based on multiple measurements of physical and biochemical properties has been developed. Cells stained with fluorescent dyes in liquid suspension enter a unique flow chamber where electrical and optical sensors measure cell volume, single- or two-color fluorescence, and light scatter, and emerge in a liquid jet that is broken into uniform droplets. Sensor signals are electronically processed several ways for optimum cell discrimination and are displayed as pulse-amplitude distributions using a pulse-height analyzer. Processed signals trigger cell sorting according to preselected parametric criteria. Sorting is accomplished by electrically charging droplets containing the cells and electrostatically deflecting them into collection vessels. This instrument is described in detail with illustrative examples of experiments using polystyrene fluorescent microspheres, cultured human cells, and human leukocytes.

A flow cytometric method has been developed that uses phase-sensitive detection to separate signals from simultaneous fluorescence emissions in cells labeled with fluorochromes having different fluorescence decay lifetimes. By CHO cells were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC). These dyes bind to DNA and protein and the fluorescence lifetimes of the bound dyes are 15.0 and 3.6 ns, respectively. Cells were analyzed as they passed through a modulated (sinusoidal) laser excitation beam. Fluorescence was measured using only a longpass filter to block scattered laser excitation light and a single photomultiplier tube detector. The fluorescence detector output signals were processed by dual-channel phase-sensitive detection electronics and the phase-resolved PIand FITC signals were displayed as frequency distribution histograms and bivariate plots. By shifting the phase of one detector channel reference signal by m/2 + degrees and the phase of the other detector channel reference signal by -m/ 2 + +z degrees, where and +z are the phase shifts associated with the PI and FITC lifetimes, the PI and FITC signals were separately resolved at their respective phase-sensitive detector outputs. This technology is also applicable to suppressing background interferences caused by cellular autofluorescence, unbound/free dye, nonspecific dye binding, and Raman and Rayleigh scattering. Key terms: Fluorescence lifetime, phasesensitive detection, propidium iodide, fluorescein isothiocyanate, fluorescence phase-resolved measurement Flow cytometry is a n important clinical diagnostic tool for use in immunology, hematology, and oncology, including its application to basic biomedical research. Clinical tests and biological experiments often require the labeling of cells with multiple fluorochromes for correlated analysis of cellular properties. A major limitation of these procedures is the availability of fluorescent dyes with a common excitation region, i.e., requiring only one excitation source, and emission spectra that are sufficiently separated to permit measurement by multicolor detection methods (18) that employ dichroic and bandpass filters. To alleviate the problem, multiple excitation sources are usually employed (17) to excite sequentially cells labeled with fluorochromes that have separated excitation spectra. This approach has greatly increased the number of fluorochromes available for multicolor labeling studies, but the instrumentation has become increasingly complex in both design and function.We have developed a new flow cytometric method (19) to separate signals from fluorochromes by phaseresolved measurement of fluorescence emission signals. Our intention is to resolve signals from fluorochromes with overlapping emission spectra, but 'Research reported in this article was performed under the auspices of the U.S. Department of Energy.