Immunofluorescent quantification of tyrosine phosphorylated proteins by flow cytometric analysis

To measure the quantity of tyrosine phosphorylated proteins in cells, we have used a flow cytometry technique with fluorescein isothiocyanate-labeled anti-phosphotyrosine antibody (FITC-PY mAb). The analysis was applied to the phosphotyrosine titration and showed an optimum amount of FITC-PY mAb (30g/1×10cells). The staining specificity of our assay was tested by the addition of exogenous competitors, showing a specific inhibition by phosphotyrosine but not by phosphoserine, phosphothreonine, or tyrosine. The assay was also able to elucidate the inhibitory effect on tyrosine phosphorylation of genistein, a protein tyrosine kinase inhibitor. These results imply that immunofluorescent quantification assay using a flow cytometer could be a useful technique to determine the intracellular level of tyrosine phosphorylated protein.     

Immunofluorescent flow cytometry in N dimensions. The multiplex labeling approach

The problem of the simultaneous use in flow cytometry of N greater than 2 antibodies in conjunction with two fluorochromes was investigated. Theoretical analysis led to a labeling procedure and reconstruction formula that allow N-dimensional labeling distributions to be obtained from two-dimensional fluorescence distributions. The general problem of M greater than or equal to 2 fluorochromes and N greater than M antibodies was shown to be reducible to the case of two fluorochromes. The method was tested by a triple labeling analysis of murine thymocytes.     

Fluorescent methotrexate labeling and flow cytometric analysis of cells containing low levels of dihydrofolate reductase

Previous studies have demonstrated the usefulness of flow cytometry in the analysis of dihydrofolate reductase (EC 1.5.1.3) gene amplification. However, this powerful and potentially sensitive method for analyzing gene expression in individual cells has not seen widespread use. This is due in part to the difficulty of producing fluorescent methotrexate (Fluo-MTX), which is needed to label dihydrofolate reductase in vivo, in yields higher than 1% and of sufficient purity to give low nonspecific backgrounds by the published procedures. We have significantly improved the synthesis of Fluo-MTX to obtain rapidly a chromatographically pure product in 20% yields. In addition, we have found that cell volume is a variable which makes direct comparisons of fluorescence intensity between cell lines difficult. In order to circumvent this problem, we have improved flow cytometric analysis to measure the fluorescence specific intensity of individual cells. A survey of various cells commonly used for gene transfer shows a significant variability in the efficiency with which they are labeled with Fluo-MTX, which appears to be due to variations in their ability to transport this reagent.     

Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis.

A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 micrograms/ml to 5 micrograms/ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed.     

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.     

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.     

New method for the analysis of flow cytometric data

A numerical method for deriving the fractions of cells in different phases of the cell cycle from a single observed DNA histogram is presented. The observed histogram is regarded as a polluted version (containing allocation errors) of the true histogram. A mathematical model is used to describe the pollution process. A theoretical histogram, representing the true histogram, is constructed so that G1 cells are put into one channel and G2M cells into another; the distribution of S cells in between is approximated with a set of harmonic functions. This theoretical histogram is subsequently disturbed with Gaussian dispersion functions to stimulate the pollution, yielding a predicted histogram. Using a maximum likelihood estimation technique, the model parameters are adjusted iteratively, matching the predicted histogram to the actually observed one. With the final parameter values substituted, the corresponding final theoretical histogram is regarded as a reliable reconstruction of the true histogram. From the latter, the required percentages can be read directly. The advantage of this approach over other mathematical analysis methods is that it allows a wide range of different, continuous distributions for relatively few model parameters (thus featuring flexibility and realism and a diminished risk of encountering computational problems). In addition, estimation errors providing a measure of accuracy can be obtained. To test the method, it was used to analyze various observed histograms from the literature that have been obtained by either simulation or actual flow cytometric measurements. The method appeared to perform well, as compared to the reported results of several other methods of analysis applied to the same data.     

Fixation procedures for flow cytometric analysis of environmental bacteria

Analysis of environmental bacteria on the single cell level often requires fixation to store the cells and to keep them in a state as near life-like as possible. Fixation procedures should furthermore counteract the increase of autofluorescence, cell clogging, and distortion of surface characteristics. Additionally, they should meet the specific fixation demands of both aerobically and anaerobically grown bacteria. A fixation method was developed based on metal solutions in combination with sodium azide. The fixation efficiencies of aluminium, barium, bismuth, cobalt, molybdenum, nickel, and tungsten salts were evaluated by flow cytometric measurement of the DNA contents as a bacterial population/community stability marker. Statistical equivalence testing was involved to permit highly reliable flow cytometric pattern evaluation. Investigations were carried out with pure cultures representing environmentally important metabolic and respiratory pathways as controls and with activated sludge as an example for highly diverse bacterial communities. A mixture of 5 mM barium chloride and nickel chloride, each and 10% sodium azide was found to be a suitable fixative for all tested bacteria. The described method provided good sample stability for at least 9 days.     

Flow cytometric double labeling technique for screening of multidrug resistance.

We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16. Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR-content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR-content were not related to differences in DNA-content. In experimental cell lines immunofluorescence data were inversely related with those of DNR-content: MDR cells had high levels of P-gp expression and low levels of DNR-content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.     

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.     

Quantitative flow cytometric analysis of ABO red cell antigens.

A flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluorescein-conjugated anti-H lectin and the A and B antigens by indirect staining first with monoclonal anti-A or anti-B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti-mouse immunoglobulin (Ig) antibodies. More than a ten-fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of greater than 200 and greater than 30, respectively, while heterozygotes (AO or BO) had ratios of less than 5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti-A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population.(ABSTRACT TRUNCATED AT 250 WORDS)     

A statistical analysis of flow cytometric determinations of phagocytosis rates.

Uptake of particles by phagocytosing cells is a process that exhibits variability of its rate. This variability is inherent in the mechanism of particle uptake and in the mechanisms that determine the distribution of physiological states within a population of phagocytosing cells. When numbers of particles ingested by cells are determined flow cytometrically an additional measurement variability is superimposed on and interacts with the aforementioned biological variability. In one method of determining population phagocytosis parameters, which involves fitting theoretical equations to experimental time course data on the fractions of cells which have ingested 0, 1, 2, ... particles, the effects of measurement variability are circumvented, although this usually has the cost of not using all the sample data obtained. However, in a second, simpler, method which is based on determining the time course of the number of particles ingested by an average cell, measurement variability is not circumvented and its effects must be considered. An analysis of the combined effects of biological and measurement variability on the results obtained with the simpler method is presented in this paper. Experimental results for phagocytosis of latex microspheres of uniform size and fluorochrome content by populations of the ciliate Tetrahymena pyriformis show that, for this system, measurement variability is entirely negligible in comparison with biological variability. This conclusion might not apply to other systems, however, and situations which might make measurement variability of some significance are mentioned in the paper. The equations given can be used for the analysis of such situations.     

Plug flow cytometry: An automated coupling device for rapid sequential flow cytometric sample analysis.

BACKGROUND: The tools for high throughput flow cytometry have been limited in part because of the requirement that the samples must flow under pressure. We describe a simple system for sampling repetitively from an open vessel. METHODS: Under computer control, the sample is loaded into a sample loop in a reciprocating eight-way valve by the action of a syringe. When the valve position is switched, the plug of sample in the sample loop is transported to the flow cytometer by a pressure-driven fluid line. By coupling the plug-forming capability to a second multi-port valve, samples can be delivered sequentially from separate vessels. RESULTS: The valve is able to deliver samples at rates ranging up to about 9 samples per minute. Each plug of sample has uniform delivery characteristics with a reproducible coefficient of variation (CV). Even at the highest sampling rate, carryover between samples is limited. CONCLUSIONS: Plug-flow flow cytometry has the potential to automate the delivery of small samples from unpressurized sources at rates compatible with many screening and assay applications.     

UV lasers for flow cytometric analysis: HeCd versus argon laser excitation.

Applying flow cytometric single cell analysis, we compared the performance of UV excitation from argon ion and HeCd lasers using various UV-excitable fluorochromes of cell kinetic and cell physiological relevance. The AT-specific DNA fluorochromes DAPI, Hoechst 33258, and Hoechst 33342 showed no significant differences of G1-phase resolution and cell cycle distribution. With the HeCd laser, high-resolution cell kinetic analysis applying the novel BrdU/Hoechst-PI quenching technique showed superior resolution and an almost normalized G2M/G1 channel ratio of the first cell cycle. Indo-1 analysis for detection of intracellular free calcium gave similar results for both excitation sources, although the indo-1 ratio of activated cells was lower for HeCd excitation. Monochlorobimane as an indicator fluorochrome of glutathione content could not be excited sufficiently with the 325-nm line of the HeCd laser and exhibited poor resolution between positive and negative cells. However, the second glutathione-specific fluorochrome o-phtalaldehyde gave even better results with the HeCd laser. Our data indicate that air-cooled HeCd lasers are cheap and reliable UV-excitation sources for most UV-excitable fluorochromes, and might be an alternative to the expensive water-cooled argon and krypton laser.     

DNA-protein flow cytometric analysis in non-Hodgkin lymphoma

A flow cytometric study of DNA and protein contents was performed on cell suspensions obtained from 73 adult patients with non-Hodgkin lymphoma. Bivariate analysis identified a second subpopulation, not revealed by DNA determination, in 25% of the tumors. Protein heterogeneity was more frequently observed in diffuse than in nodular histology according the Rappaport classification and in high-grade than in low-grade malignancy tumors by the Kiel classification and the Working Formulation, but it was not related to ploidy or cell proliferative rate. The presence of an additional subpopulation, detected by protein analysis, defined as monoclonal by DNA analysis, could adversely affect clinical outcome in terms of response to treatment and overall survival.     

DNA flow cytometric analysis of mouse seminiferous epithelium

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.     

Enzymatic amplification staining for flow cytometric analysis of cell surface molecules

BACKGROUND: Flow cytometric analysis is a powerful technique for the single cell assessment of cell surface expression of selected molecules. The major deficiency of flow cytometry has been its relative insensitivity. Only molecules expressed in abundance have been readily observed. METHODS: We have developed an enzymatic amplification procedure for the analysis of cell surface molecules by flow cytometry. Transformed and nontransformed cells expressing MHC class I, CD5, CD3, CD4, CD6, CD7, CD34, CD45, MHC class II, Fas ligand, and phosphatidylserine were assessed. RESULTS: Our enzymatic amplification technology increased the fluorescence signal between 10 and 100-fold for all surface molecules tested. CONCLUSIONS: Enzymatic amplification staining produces a significant enhancement in the resolving power of flow cytometric analysis of cell surface molecules. Using this technique, we have been able to detect the presence of molecules that could not be observed by the standard procedure.