Quantitative analysis of a nuclear antigen in interphase and mitotic cells

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.     

Simultaneous detection of cyclin B1, p105, and DNA content provides complete cell cycle phase fraction analysis of cells that endoreduplicate

BACKGROUND: DNA analysis of endoreduplicating cells is difficult because of the overlap between stem-line G2 + M cells and 4C G1 cells. Simultaneous flow cytometry of DNA and cyclin B1 analytically separates these populations. The objective here was to develop simultaneous flow cytometry of DNA, cyclin B1, and p105 (highly expressed in mitosis) for improved, complete cell cycle phase fraction analysis of endoreduplicating cell populations. METHODS: Monoclonal antibody, GNS-1, reactive with human cyclin B1, was conjugated with fluorescein at three different fluorochrome-to-protein (F/P) ratios and tested for optimal sensitivity in a flow cytometric assay. A formaldehyde-methanol fixation procedure was optimized for retention of p105 within mitotic cells by analytic titration of formaldehyde. p105 was stained indirectly with Cy5-conjugated secondary antibody, followed by GNS-1, and DNA was stained with Hoechst 33342. The specificity of p105 in this assay was tested by comparison of manual and flow cytometric mitotic indices and by sorting and microscopic inspection. RESULTS: F/P 4.1 provided optimal fluorescein labeling of GNS-1. Formaldehyde (0.5%), followed by methanol permeabilization, fixed cells sufficiently to quantify stem-line and endoreduplicated G1, S, G2, and M phase fractions. Kinetic measurements of these fractions for both populations were demonstrated. CONCLUSIONS: The fluorochrome-to-protein ratio is important and can be optimized objectively for these assays. A permeabilization-sensitive antigen (p105), previously requiring formaldehyde/detergent-fixed cell preparations, was shown to work equally well with formaldehyde/ methanol fixation. Three-laser, two-parameter intracellular antigen analysis can be successfully coupled with DNA content analysis. Cell cycle kinetic analysis of endoreduplicating populations should be improved.     

A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry

A preparative technique for the two-parameter flow cytometric study of nuclear antigen expression is reported. This method employs a brief sequential treatment of cells at 4 degrees C first with 0.5% paraformaldehyde and second with 0.1% Triton X-100 in phosphate-buffered saline followed by cellular staining with indirect immunofluorescence and propidium iodide. Using this technique, cellular morphology is preserved, cell clumping is minimized, and high-quality indirect immunofluorescence and DNA staining are obtained with a minimum of nonspecific labeling. Utilizing nuclear antigen-specific monoclonal antibodies in conjunction with this technique, the cell-cycle phase-dependent expression of such antigens is examined. From these data, the utility of two-parameter flow cytometry in the identification and quantification of cell-cycle-dependent modulation of nuclear antigens is discussed.     

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.     

Identification of a nuclear protein component of interchromatin granules using a monoclonal antibody and immunogold electron microscopy

A monoclonal antibody, designated 780-3, has been generated which preferentially recognizes an antigenic component of interchromatin granules in human cells. By indirect immunofluorescence procedures, monoclonal antibody 780-3 produces a cell cycle-specific speckled nuclear staining pattern in adult human fibroblasts which is dramatically altered during metaphase. In contrast, transformed cells appear to express this antigen throughout the cell cycle in increased quantities. Immunogold electron microscopy revealed that the nuclear antigen is intimately associated with interchromatin granules in human cells. Analysis by immunoblot procedures showed that monoclonal antibody 780-3 recognizes two polypeptides of 105 and 41 kD. From these data, a possible nucleolar derivation of interchromatin granules is discussed. These studies demonstrate for the first time that monoclonal antibodies may be used in combination with immunogold electron microscopy to identify the ultrastructural location of nuclear antigens.     

Establishment of a lymphoblastoid cell line and isolation of an Epstein-Barr-related virus of gorilla origin.

A B-lymphoid cell line was established from a normal gorilla. The cells contained Epstein-Barr virus-related antigens, and herpesvirus particles were demonstrated by electron microscopy. DNA-DNA reassociation kinétics revealed 30 to 40% hybridization to Epstein-Barr virus with 50 genomes per cell. Examination of the viral nuclear antigen with gorilla sera showed this to be a unique isolate termed Herpesvirus gorilla. H. gorilla transformed gibbon B-lymphocytes in vitro.     

Flow cytometric analysis of estrogen, progesterone receptor expression and DNA content in formalin-fixed, paraffin-embedded human breast tumors.

Flow cytometric analysis of estrogen (ER) and progesterone (PgR) receptor expression in archival human breast tumors is relatively difficult. We have used enzyme digestion and microwave antigen retrieval procedures for multiparametric flow cytometric analysis of ER and PgR expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded primary breast tumors. Deparaffinized rehydrated tissue sections treated with pepsin were subjected to microwave irradiation for unmasking of ER and PgR antigenic sites. Biotinylated ER antibody and streptavidin-fluorescein isothiocyanate (FITC) were used for ER labeling and PgR antibody with phycoerythrin labeled goat anti-mouse antibody was used for PgR labeling. Counter staining with propidium iodide-RNase was used for determination of cellular DNA content. Our results show that enzyme digestion and microwave treatment of formalin-fixed, paraffin-embedded breast tumors can be successfully used for the multiparametric analysis of nuclear hormone receptor expression and DNA content by flow cytometry.     

Analysis of c-erbB-2 protein expression in conjunction with DNA content using multiparameter flow cytometry

Breast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c-erbB-2 protooncogene is amplified in 10-40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c-erbB-1 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c-erbB-2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c-erbB-2 expression in populations of ethanol-fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c-erbB-2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.     

In vivo immunogold labeling confirms large-scale chromatin folding motifs

The difficulty in localizing specific cellular proteins by immuno-electron microscopy techniques limits applications of electron microscopy to cell biology. We found that in vivo immunogold labeling improves epitope accessibility, ultrastructural preservation and three-dimensional visualization, and allows correlated light and electron microscopy. We detected large-scale chromatin folding motifs within intact interphase nuclei of CHO cells and visualized the ultrastructure of DNA replication 'factories' labeled with GFP-proliferating cell nuclear antigen (PCNA).     

Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells

We have investigated the effects of ceramide on the progression of cell cycle and on apoptotic cell death in ovarian cultured granulosa cells. Rates of cellular proliferation were measured by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and flow cytometric cell cycle analysis. We also examined for morphological and biochemical signs of apoptosis. The PCNA expression was downregulated in a dose-dependent manner after treatment with C6-ceramide. Flow cytometric analysis demonstrated that the exposure of granulosa cells to C6-ceramide markedly decreased the population associated with G0/G1 DNA content and the reduction of cell numbers in G0/G1 phase was accompanied by the elevation of the A0 phase. The exposure of granulosa cells to exogenous C6-ceramide induced drastic morphological changes including cytoplasmic- or nuclear condensation and typical apoptotic DNA degradation. We also observed that phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, significantly inhibited the ceramide-induced apoptosis. These results suggested that ceramide might block the progression of cell cycle at G0/G1 phase and as a consequence, granulosa cells would be committed to apoptosis. Our findings also indicated that down-regulation of the PKC activity might be involved in the ceramide-induced apoptosis in cultured granulosa cells.     

Flow Cytometry of Mitotic Cells

We have developed a procedure using flow cytometric measurement of a mitosis-specific antigen that may be used to count mitotic cells and sort them from nonmitotic cells. The procedure may also be used in conjunction with measurement of cellular DNA content and of bromodeoxyuridine incorporation into cellular DNA to assign cells to the G1/G0, S, G2, or M phase of the cell cycle.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36–40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G₂/M phase of the cell cycle was significantly higher than that in G₀/G₁ phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G₂/M phase.     

Analysis of apoptosis by propidium iodide staining and flow cytometry

Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories.     

Growth kinetics as a function of ploidy in diploid, tetraploid, and octaploid smooth muscle cells derived from the normal rat aorta

The smooth muscle cell population in major arteries of humans and experimental animals is heterogeneous with regard to cellular DNA content. A proportion of cells has polyploid DNA content and this proportion increases with normal aging and with hypertension. We have isolated pure populations of rat aortic smooth muscle cells containing 2C, 4C, and 8C DNA content by cloning of cultures of cells previously subjected to flow cytometric cell sorting. Karyologic analysis of these clonal populations revealed them to be pure diploid, tetraploid, and octaploid populations, respectively, containing 2N (= 42), 4N, and 8N chromosomes. Cell attachment area and nuclear size appeared to increase with the level of ploidy. Studies of the proliferative characteristics of the cells revealed that the growth rate and ultimate cell densities achieved decreased as the ploidy level increased. The intrinsic cellular radiosensitivity of these clones did not vary with ploidy. Increased smooth muscle cell ploidy is, therefore, associated with a decreased rate of proliferation. The emergence of smooth muscle cells with polyploid DNA content under normal and pathologic conditions is probably due to mitotic polyploidization without net cell proliferation and may be related to the need for expression of differentiated functions.     

Early cellular events during induction of carrot explants with 2,4-D

Summary The cellular events occurring in carrot hypocotyl explants during long-term and pulse treatment with 2,4-D were followed using different techniques (light and transmission electron microscopy, flow cytometry, PCNA staining). Different morphogenetic pathways were induced under the various experimental conditions. Nevertheless, in the explants the activated cells were the same (provascular cells) and they showed very similar structural and ultrastructural changes. The long-term treatment with 2,4-D induced rapid re-activation of the cell cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - PCNA proliferating cell nuclear antigen - ER endoplasmic reticulum - GA Golgi apparatus     

Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses

The morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X- and Y-chromosome-bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate-buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′-6-diamidino-2-phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high-resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.     

Investigation of the effectiveness of disinfectants against planktonic and biofilm forms of P. aeruginosa and E. faecalis cells using a compilation of cultivation,microscopic and flow cytometric techniques

This study evaluated the effectiveness of selected disinfectants against bacterial cells within a biofilm using flow cytometry, the conventional total viable count test and scanning electron microscopy (SEM). A flow cytometric procedure based on measurement of the cellular redox potential (CRP) was demonstrated to have potential for the rapid evaluation of activity against biofilm and planktonic forms of microbes. Quaternary ammonium compound-based disinfectant (QACB) demonstrated a higher level of anti-microbial activity than a performic acid preparation (PAP), with mean CRP values against P. aeruginosa cells of 2 and 1.33 relative fluorescence units (RFU) vs 63.33 and 61.33 RFU for 8 and 24 h cultures respectively. Flow cytometric evaluation of the anti-biofilm activity demonstrated a higher efficacy of QACB compared to PAP for P. aeruginosa cells of 1 and 0.66 RFU vs 18.33 and 22.66 RFU for 8 and 24 h cultures respectively. SEM images of treated P. aeruginosa cells demonstrated disinfectant-specific effects on cell morphology.     

Trophectoderm surface expression of the cell adhesion molecule cell-CAM 105 on rat blastocysts

A variety of cellular interactions is involved in the process of implantation of the mammalian embryo into the uterine tissue. Recent discoveries have demonstrated that intercellular recognition and adhesive events are governed by a class of cell surface molecules known as cell adhesion molecules (CAMs). In the present report, we have investigated the occurrence of the well-characterized cell adhesion molecule cell-CAM 105 on the surface of rat pre- and peri-implantation embryos of various stages. This was carried out by indirect immunofluorescence microscopy employing affinity-purified rabbit antibodies against cell-CAM 105. The embryonal stages investigated comprised morulae, normal day-4 blastocysts, and delayed and adhesive blastocysts obtained by using the method of experimentally delayed implantation. Cell-CAM 105 was absent in the early-morula stage, but in normal day-4 blastocysts and delayed blastocysts a specific staining for cell-CAM 105 was seen on the entire surface. However, adhesive-stage blastocysts exhibited a marked polarity with staining of the polar trophoblast cells. Scanning electron microscopy of adhesive-stage blastocysts revealed that the stronger staining of the polar region was not due to a greater number of microvilli on the polar trophoblast cells. Thus, it seems as if cell-CAM 105 is lost or masked from the surface of the mural trophoblast cells of adhesive-stage rat blastocysts. Since the mural trophoblast cells are the first to adhere to the uterine luminal epithelium during the onset of implantation and subsequently invade the uterine stroma, we suggest that the apparent downregulation of cell-CAM 105 in the mural trophoblast cells might be linked to the acquisition of trophoblast invasiveness.