Simultaneous flow cytometric analysis of two cell surface markers,telomere length,and DNA content

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.     

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.     

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.     

Multiparameter flow cytometric approach for simultaneous evaluation of T lymphocyte-endothelial cell interactions.

Since vascular endothelium is now recognized as an immunologically active tissue, a better understanding of the relationship between endothelial cells and T lymphocytes is critical to the field of solid organ transplantation. Investigations of endothelial cell-T cell interactions have been limited by methodology. We developed a flow cytometric method allowing for concurrent investigation of multiple cell populations within the same culture that can be applied to these complex interactions. Allogeneic CD8+ or CD4+ T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycle-blocking agent. At specific time points, the coculture was analyzed by flow cytometry. T-cell proliferation could be detected by gating on the T-cell subset and evaluating the CFSE fluorescence peaks. By directly analyzing cellular division, we minimized erroneous interpretation of the data encountered by previous studies, which utilized (3)H-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilitated the study of CD8+ lymphocyte activation, differentiation, and acquisition of effector function. By gating on the endothelial cell population, phenotypic changes such as upregulation of surface MHC molecules or immune-mediated apoptosis could be detected. In conclusion, we present a flow cytometric approach that could have important applications for clinical immunological monitoring in allogeneic or xenogeneic transplantation, and might provide the requisite information to better tailor immunotherapy to prevent chronic rejection.     

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.     

Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327-G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA-stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H-TdR, colcemid, or hydroxyurea + colcemid showed that 1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, 2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and 3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build-up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H-TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid-blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content. The nuclear DNA/protein histograms of untreated and colcemid-treated log-phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen-sensitive rat prostatic R3327-G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.     

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.     

Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.     

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.     

A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.     

A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification.

A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4 degrees C with 0.25% buffered paraformaldehyde then for 15 min at 37 degrees C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1-peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90 degrees angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual-color surface antigen staining combined with DNA staining with 7-amino-actinomycin D (7-AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF-CEM for expression of CD3 epsilon and on NALM-6 for expression of mu. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.     

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)     

Technical and statistical improvements for flow cytometric DNA analysis of paraffin-embedded tissue

Flow cytometric DNA analysis of paraffin-embedded solid tumors has permitted review of large series of archival tissue in attempts to relate abnormal DNA content to prognosis. Limitations of the technique include: 1) a laborious, time-consuming procedure; 2) variation in technique between laboratories; and 3) lack of an objective method of computing DNA indices. Critical evaluation of our technique has shortened the time involved in dewaxing and rehydration, selectively utilized patient's own normal tissue as the internal standard, proved reproducibility of stored specimens, standardized DNA index computation, and developed a statistical analysis to confirm aneuploidy. These technical improvements and the development of a statistical analysis provide a way to shorten the procedure time and standardize the data generated from flow cytometric DNA analysis so as to improve the quality of retrospective reviews of paraffin-embedded tumors and accelerate the definition of flow cytometry's role as a prognostic indicator.     

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation.

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.     

DNA mapping of gastric cancers using flow cytometric analysis

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.     

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.     

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.     

Immunofluorescent labeling of centromeres for flow cytometric analysis.

A procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody-labeled human and hamster chromosomes were 4-10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.     

Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry

Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.