Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability.

BACKGROUND: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. MATERIALS AND METHODS: Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. RESULTS: Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD-negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. CONCLUSIONS: The results show that cells stained with FITC-labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions.     

Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.     

Assessment of cell viability by flow cytometric analysis using DNase exclusion

A new method was developed for selective measurement of DNA distributions in viable cell populations. The method is based on the fact that non-viable cells lose membrane integrity and treatment of such cells with DNase should remove their DNA. The DNase-treated cells were stained with DNA fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) in the presence of Triton X-100. DNA distribution was measured by flow cytometry prior to and after treatment with DNase. Percentage of cells stained after DNase treatment was considered as an index of cell viability. Optimal conditions for DNase treatment and application of DNase exclusion test for the analysis of spontaneous cell death, selective death of cells arrested in S/G2 phases, instant cell disintegration induced by cytotoxic compounds and cell death induced by hyperthermia are described.     

Rapid flow cytometric method for viability determination of solventogenic clostridia

We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone–butanol–ethanol (ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains, BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability. Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination. We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation.     

Use of monoclonal antibodies for flow cytometric detection of intracellular Toxoplasma gondii in murine splenic lymphocytes

Various monoclonal antibodies (mAbs) against Toxoplasma gondii RH tachyzoites were used for flow cytometric detection of intracellular parasites in murine splenic lymphocytes. Tg110 and Tg563 (reacting with the major surface protein SAG1), Tg505 (with another surface protein SAG2), Tg695 and Tg786 (with rhoptry proteins), Tg507, Tg621, and Tg317 (with dense granule proteins), Tg536 (with a microneme protein), and Tg685 (with a cytosol antigen) were the mAbs used. After an in vitro infection of lymphocytes with tachyzoites and reactions with the different mAbs, flow cytometry was performed using an indirect immunofluorescent technique. The proportions of whole infected lymphocytes and of each infected lymphocyte phenotype, CD4+ T cells, CD8+ T cells, and B cells, were determined, and their fluorescent intensities were quantified. The best reaction was seen when Tg110 or Tg695 was used as the mAbs. The results suggest that mAbs against surface or rhoptry proteins are highly useful for the flow cytometric detection of intracellular T. gondii in host cells.     

Increased apoptosis of adult rat lymphocytes after single neonatal vitamin a treatment (hormonal imprinting). A flow cytometric analysis

Newborn rats were treated with a single dose of vitamin A (retinol) and apoptosis of peripheral lymphocytes was studied by flow cytometry in adult age. Vitamin A treatment (hormonal imprinting) caused a moderate, however significant elevation in the number of apoptotic lymphocytes after three months. Dexamethasone or Concanavalin-A alone did not influence apoptosis significantly. However, in the neonatally retinol treated rats dexamathasone significantly elevated the quantity of apoptotic lymphocytes related to the control or Concanavalin-A treated control cells. The results call attention to the prolonged effect of hormonal imprinting in a new index and to the possible dangerous effects in human, neonatally treated with vitamin A.     

A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white M2R

Analysis of dead versus live cells is shown to be possible using Calcoflour White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two-color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.     

Comprehensive quality assessment approach for flow cytometric immunophenotyping of human lymphocytes

Flow cytometric immunophenotypic (IPT) evaluation has become an important adjunct to clinical patient management and epidemiological studies. This has precipitated a need for stringent quality assessment (QA) procedures to ascertain data integrity. We evaluated a QA approach to monitor all elements of the immunophenotyping process, inclusive of blood collection and processing procedures as well as of staining reagent and instrument performance. Central to our approach was preparation each day, in parallel with clinical analytes, of lymphocytes from healthy donors, selected from a 15 donor panel. IPT parameters evaluated over a 19 month period included frequencies of CD3+, CD4+, CD8+, and CD20+ lymphocytes and the ratio of CD4+ to CD8+ lymphocytes. The sensitivity for analytical error detection was reflected by median coefficients of variation of these parameters within individual panel donors, which were 4.1%, 4.5%, 3.9%, 8.2%, and 10.1%, respectively. IPT parameter values were determined each day for two of the panel donors, then averaged and standardized to obtain a quality or Q variate, which was the basis of QA. Error detection sensitivity decreased 0.6-1.7% and the number of false rejections increased 1.2-3.3% when one panel donor rather than two was used daily for QA. This study also illuminated important aspects of what constitutes the norm for longitudinal IPT parameter variation in healthy individuals including: 1) a generally low degree of temporal parameter variation within individual donors, but 2) significant differences between donors with respect to variance estimates for CD3+ and CD8+ lymphocyte frequencies and CD4+/CD8+ lymphocyte ratios, and 3) an apparent seasonal pattern of variation in CD4+ T-cell frequencies.     

A low-cost flow cytometric assay for the detection and quantification of apoptosis using an anionic halogenated fluorescein dye

We describe here a technical improvement of an established colorimetric method used to detect and measure the occurrence of apoptosis in mammalian cells during in vitro cell culture. This assay uses an anionic halogenated fluorescein dye that is taken up by apoptotic cells at the stage of phosphatidylserine externalization. We demonstrate that apoptotic cells stained with this dye can be detected by flow cytometric analysis. Furthermore, we show that the modified method compares well with the standard annexin-V-based apoptosis assay and that it is significantly more cost-effective than the annexin-V assay.     

Simultaneous cell surface phenotype and cell cycle analysis of lymphocytes by flow cytometry

A method for the simultaneous measurement of cell surface components and nucleic acids (DNA and RNA) of human lymphocytes by flow cytometry has been developed, thereby providing a means of analyzing cell surface changes during the various phases of the cell cycle. Unfixed cells were coated with fluorescein-conjugated concanavalin A (F Con A) or surface antigen-specific antibody, fixed sequentially with paraformaldehyde and methanol, treated with specific nucleases, and then stained with propidium iodide. Neither portion of the procedure (cell surface staining, nucleic acid staining) interfered significantly with the other. Cell cycle phases of phytohemagglutinin-stimulated human lymphocytes as determined by this method were comparable with those identified by acridine orange staining. Cell cycle-specific blocking agents were used to additionally demonstrate the specificity of the staining procedure. Simultaneous measurement of cell cycle phase and detection of surface receptors for Con A and T lymphocyte surface determinants was performed with this method.     

Preparation of tissues for DNA flow cytometric analysis

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4',6-diamidino-2 phenylindole-2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5-10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.     

Intrinsic properties of so-called dormant probiotic bacteria, determined by flow cytometric viability assays

Plate counting and four culture-independent flow cytometric assays were used to determine the viability and intrinsic properties of three probiotic strains during storage. The strains showed reduction in plate counts but were able to maintain esterase activity, intact cytoplasmic membrane, and pH gradient. The apparently uncultivable probiotic cells were active and stress resistant.     

Method for flow cytometric detection of Listeria monocytogenes in milk

This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures.     

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum.

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2–3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.     

A simple preservative for flow cytometric DNA analysis

A solution containing citric acid buffered saline (CABS) and 99% ethanol (E) 1:1 was used for preserving cells for flow cytometric DNA analysis. DNA histograms obtained from fine needle biopsy aspirates and preserved in CABS+E had a similar mean coefficient of variation (CV) as was obtained from aspirates taken in CABS (3.3 vs. 3.4%) and a clearly smaller mean CV than was obtained from aspirates preserved in 50% ethanol (mean 4.8%, P less than .0001). Aspirates taken in CABS more often contained a small (less than 3,000) number of cells as compared with aspirates preserved either in CABS+E or ethanol (P less than .0001). Since preservation of cells in CABS+E allows long-term storage of samples and results in a decreased number of insufficient samples as compared with buffered saline and in an enhanced resolution as compared with 50% ethanol, CABS+E is recommended for preservation of cytological samples to be analyzed for DNA content with flow cytometry.     

Cell-by-cell flow cytometric analysis of chromosomes

The authors discuss various aspects of a recently developed method permitting a detailed flow cytometric analysis of the individual cell karyotypes such as instrumentation, histochemistry, data proceeding algorithms. Possible drawbacks of the method and the ways of their overcoming are considered. Results of analysis of the Chinese hamster cells are presented that illustrate the possibilities of the method, including the metaphase chromosome distribution according to their fluorescence intensity, the analysed cell distribution according to their chromosomes number, the table in which the individual cell karyotypes are distributed according to their fluorescence. The results obtained show that the developed method may be successfully used for investigating chromosomal iNstability and heterogeneity of the mammalian cells.     

Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl-positive cells.

BACKGROUND: Constitutive tyrosine phosphorylation derived from Bcr-Abl kinase activity is the major characteristic of Bcr-Abl positive cells. In this study, we developed a method to detect the phosphotyrosine proteins by flow cytometry and we asked whether phosphorylation was affected by imatinib mesylate treatment. METHODS: Cells were treated or not with imatinib mesylate, fixed and permeabilized by PFA followed by saponin, then stained with anti-phosphotyrosine (p-tyr) monoclonal antibody and analyzed by flow cytometry. RESULTS: Optimal staining parameters were performed with p-tyr antibody using K562 and LAMA84 lines that displayed high levels of tyrosine phosphorylation as compared to the control line, HL60. Tyrosine phosphorylation was inhibited by imatinib in a dose-dependent manner, but not modified by other inhibitors demonstrating that the staining detected is specific to Bcr-Abl phosphorylation. The staining of imatinib-resistant cell lines such as the mutated BaF/Bcr-AblT315I cell line or resistant CML patient cells, showed that hyperphosphorylation was not affected by imatinib treatment. In one CML patient, our technique permitted us to detect a small hyperphosphorylated population resistant to imatinib that appeared hyperphosphorylated and amplified at the time of relapse. CONCLUSIONS: We have developed a flow cytometric technique presenting several advantages such as rapidity and sensitivity, which requires fewer cells than the Western blot.     

Flow cytometric analysis of opposite effects of a monokine on proliferation and differentiation of murine B lymphocytes.

A 35,000 mw factor able to replace macrophages (FRM) in the induction of the in vitro antibody response to sheep erythrocytes has been isolated from the supernatant of murine resident peritoneal macrophage cultures. Purified FRM, when added at the outset of cultures, induced B cells to generate an antigen-specific antibody response. The signals provided by FRM in the process of B cell activation were analyzed using a polyclonal model. Cell cycle analysis by multiparameter flow cytometry after acridine orange staining showed that FRM, on its own, did not trigger the transition of B cells from the G0 to the G1 stage of the cell cycle. In addition, FRM affected neither the basal intracellular free calcium level ([Ca2+]i) nor the rapid increase in [Ca2+]i induced by crosslinking of membrane immunoglobulin (mIgM) with anti-mu antibodies. In parallel with its positive effect on B cell differentiation, FRM markedly reduced both proliferation and cell cycle progression of B cells stimulated with anti-mu plus interleukin 4 (IL-4). Indeed, the addition of FRM to such cultures led to a preferential accumulation of cells in the early G1 compartment of the cell cycle and to a decreased frequency of cells in all other phases including G1B, S and G2/M.     

Simultaneous flow cytometric detection of cellular c-myc protein, incorporated bromodeoxyuridine, and DNA

We describe a multivariate flow cytometric technique for simultaneous analysis of specific nuclear protein, bromodeoxyuridine (BrdUrd) incorporated into DNA and DNA content in single cells in suspension. The procedure involves fixation of BrdUrd-exposed cells with paraformaldehyde, heat denaturation of cellular DNA, followed by sequential immunochemical reactions to label incorporated BrdUrd and nuclear protein, and finally staining of total DNA with propidium iodide. The cells are analyzed flow cytometrically and multivariate data acquired in list mode to facilitate analyses of heterogeneous subpopulations. We applied this technique to measure c-myc protein, incorporated BrdUrd, and DNA content in subpopulations present in a recombinant Chinese hamster ovary (CHO) cell line carrying approximately 800 copies of murine c-myc sequences under control of an inducible heat shock promoter.     

Identification of deficient mitochondrial signaling in apoptosis resistant leukemia cells by flow cytometric analysis of intracellular cytochrome c, caspase-3 and apoptosis

Deficient activation of apoptosis signaling pathways may be responsible for treatment failure of malignant diseases. In primary leukemia samples the detection of deficient mitochondrial apoptosis signaling would enable identification of chemo-resistant cells. To investigate the key events of apoptosis at the mitochondrial level, we developed a flow cytometric method for simultaneous detection of mitochondrial cytochrome c release and caspase-3 processing using conformation sensitive monoclonal antibodies. This method proved to identify deficient mitochondrial apoptosis signaling in leukemia cells overexpressing Bcl-2 by a pattern of apoptosis resistance, deficient cytochrome c reduction and partial processing of caspase-3. In primary leukemia cells, reduction of cytochrome c and caspase-3 activation was induced by treatment with anticancer drugs in vitro. In leukemia cells of a patient with resistant disease, a pattern of deficient apoptosis signaling as in Bcl-2 transfected cells was observed, suggesting that deficient mitochondrial signaling contributed to the clinical phenotype of drug resistance in this patient. Flow cytometric analysis of mitochondrial apoptosis signaling may provide a useful tool for the prediction of drug resistance and treatment failure in primary leukemia.