An improved method to investigate staining kinetics in single cells

A new method to analyze staining processes in single cells of histochemical and cytochemical specimens in situ is described. The combination of a microscope photometer with a perfusion cuvette developed in our laboratory allows the continuous observation of a cell during the staining process. The flow rate dependence of the staining process has been examined demonstrating the strong suppression of the diffusional boundary layer adjacent to the cell surface by sufficiently high flow rates. Experiments to find optimal conditions for the kinetic analysis of the staining reaction of nuclei in lymphocytes, neutrophile granulocytes and monkey kidney cells with thionin are described. Half-staining times of the binding of monomer dye molecules and aggregates to nuclei have been calculated; they depend on the pretreatment of the cells. The addition of electrolytes decreases the rate of staining. The formation of aggregates obeys approximately a first-order reaction law and the binding of monomers provides an order of reaction of n = 0.5.     

Staining kinetics in single cells

Summary Applying hydrodynamic conditions, which certify a negligible influence of convective diffusion, the time-dependent uptake of thionin in lymphocytes, monkey kidney cells, and their separated nuclei was measured spectroscopically. Using fixed cell material the dye transport inside the cell is not hindered due to plasma membrane and cytoplasm. The staining rate depends on the dye concentration, the pretreatment of the cell, and on the electrolyte concentration of the dye solution. The mechanism of dye migration inside the cell is in accordance with a porous matrix model. The diffusion process takes place inside the pores and channels filled with liquid and is modified by adsorption of dye molecules on the walls of the pores. A dynamic reversible equilibrium exists between migrating dye molecules and the binding sites on the pore walls described by the Freundlich adsorption isotherm. The proposed model explains the observed order of reaction of the staining kinetics.     

Staining kinetics in single cells. Part II. Diffusion processes inside the cell

Applying hydrodynamic conditions, which certify a negligible influence of convective diffusion, the time-dependent uptake of thionin in lymphocytes, monkey kidney cells, and their separated nuclei was measured spectroscopically. Using fixed cell material the dye transport inside the cell is not hindered due to plasma membrane and cytoplasm. The staining rate depends on the dye concentration, the pretreatment of the cell, and on the electrolyte concentration of the dye solution. The mechanism of dye migration inside the cell is in accordance with a porous matrix model. The diffusion process takes place inside the pores and channels filled with liquid and is modified by adsorption of dye molecules on the walls of the pores. A dynamic reversible equilibrium exists between migrating dye molecules and the binding sites on the pore walls described by the Freundlich adsorption isotherm. The proposed model explains the observed order of reaction of the staining kinetics.     

Cytological Analysis of Anastomoses and Vegetative Incompatibility Reactions in <Emphasis Type="Italic">Helicobasidium monpa</Emphasis>

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus. Received: 13 June 2001 / Accepted: 8 August 2001     

Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei.

A one-step procedure for the preparation of nuclei for flow microfluorometric DNA analysis is described. The membranes of the cells were lysed by the non-ionic detergent Nonidet P40. Single-cell suspensions, and specimens of solid tissues obtained with fine-needle biopsy, could be prepared equally well as the nuclei of solid tissue cells were released separately. Lysis was performed in the staining solution containing either ethidium bromide or propidium iodide. Fluorescence due to fluorochrome binding to RNA, was abolished instantaneously by the presence of RNA-se, and fluorochrome binding to secondary binding sites in DNA was inhibited with NaCl. The preparation time was 10 min and the samples were stable for a minimum of 12 h. With the basic version of the method, usable, but not always optimal, results were obtained in all the cell types tested: four different mouse ascites tumors, leucocytes, bone-marrow, liver cells, human lymphomas, human carcinomas of the breast and lung, mouse mammary carcinoma and solid JB-1 tumor. The method was further optimized for the JB-1 ascites tumour. The resulting two modified techniques are described. Differences in the staining of leucocytes with the analogues ethidium bromide and propidium iodide were demonstrated.     

Effect of inoculum on yeast cell aggregation

Summary Saccharomyces uvarum, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Candida sp. were induced to form cell aggregates in a column. The conditions for this induction were high cell density and slow flow rate; with K. marxianus and Candida sp., the presence of ethanol in the growth medium was also required.When these aggregated cells were inoculated into a fresh growth medium, the ability of the progeny cells to aggregate depended on the state of the inoculum. If the aggregates were not disrupted, the progeny cells remained as aggregates, and if the aggregates were dispersed by vortexing before inoculation, the progeny cells because a free cell suspension.     

Multiple nucleoli and enhanced nucleolar activity in the nurse cells of the insect ovary

Origin and function of the nucleolar apparatus in nurse cell nuclei of Calliphora erythrocephala have been investigated by cytological and autoradiographic methods in some inbred lines of laboratory blowflies with well paired polytene chromosomes in the nurse cell nuclei. Besides the nucleolus at chromosome VI large numbers of multiple free nucleoli develop in the highly polyploidized nurse cells during oocyte growth. The nucleoli incorporate H³-uridine in a considerable amount producing a homogeneous and RNase-sensitive label even after short time incubation. Their capacity of RNA synthesis is independent of their spatial relationships to other nuclear components. DNA particles in the nucleoli could be identified by the Feulgen reaction and by fluorescence staining with N,N'-diethylpseudoisocya-ninchloride, which also demonstrates the existence of own templates for autonomous RNA synthesis. There are indications that the nucleolus' own DNA is produced by gene amplification beyond the level of endomitotic polyploidization in the nurse cell nuclei. A quantitative estimation of grain density in the autoradiograms shows a rigorous shift of rRNA synthesis: at least 72% of all newly synthesized macromolecular RNA in nurse cell nuclei as contrasted to 13 % of nucleolar RNA synthesis in bristle forming cells with a similar degree of polyploidy. It seems that the nurse cell nuclei of Calliphora in addition to polyploidization increase their template capacity for synthesizing rRNA in a similar way as has repeatedly been demonstrated for Amphibia. Cytological and physiological peculiarities of the nurse cells have been discussed from the viewpoint of their functional similarity to the oocyte nucleus.     

A device for sample pretreatment during flow cytometry]

A device for the preliminary treatment of samples immediately prior to flow cytofluorimetric analysis is described. The device is intended for several procedures: (a) mixing of batched sample volumes with the reagent and efficient stirring of the mixture; (b) disintegration of cell aggregates; and (c) disruption of cell membranes to release the cell contents (chromosomes, micronuclei, nuclei etc.). The pretreatment is useful for studying the kinetic parameters of fast cellular processes in the flow, a more correct analysis of the cell cycle and the study of karyotypes of single mitotic cells. The device was called a magnetic microstirrer.     

Some observations on the mechanisms of blocking of nuclear staining by cisplatin

Summary The effect of cisplatin (cis-dichloro-diamminoplatinum II) treatment on staining of nuclei with various basic dyes and with the Feulgen reaction has been studied. Although cisplatin is reported to show negligible reaction with DNA phosphates, it has a substantial blocking effect on staining with most dyes. Short treatment with cisplatin results in binding mainly to guanine bases of DNA, causing partial blocking of the Feulgen reaction and almost complete blocking of ethidium intercalation; binding of neutral red and crystal violet is enhanced, apparently as a result of cisplatin-induced denaturation of DNA. Very prolonged cisplatin treatment does not completely block the Feulgen reaction, indicating that reaction of cisplatin with purine bases is not complete. Since attachment of cisplatin to DNA bases is unlikely to prevent binding of most basic dyes, it is suggested that the blocking of their staining may result from steric hindrance caused by formation of DNA-protein cross-links by cisplatin. Whatever the mechanism, it is incapable of producing complete blocking of staining with certain dyes. As a practical tool, it appears that rapid and almost complete blocking of staining by cisplatin may be used as an indicator of intercalative binding of dyes to DNA.     

An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry

A new staining protocol is described for the immunocytochemical detection of BrdUrd labeled nuclei. Pepsin treatment of ethanol fixed cells or tissue, followed by DNA denaturation at low pH, resulted in increased sensitivity of BrdUrd staining comparable to the thermal denaturation protocol, and decreased background binding. This technique is applicable to cell suspensions, including cultured cells and bone marrow cells. Furthermore, pepsin digestion of ethanol fixed tissue fragments resulted in a high recovery of nuclei in which incorporated BrdUrd could be detected. This possibility, together with the high sensitivity, make this method especially suitable for cell kinetic studies of human solid tumors in vivo.     

Development of an optical method for monitoring protoplast formation from cultured plant cells

The size distribution of cell aggregates during protoplast isolation from Catharanthus roseus and Nicotiana tabacum was measured by a Coulter counter. It was observed that a gradual reduction in the size of cell aggregates occured during protoplast formation. A previously developed specialized spectrophotometer for the photometric measurement of plant cell concentration was used for continuous monitoring of the reduction in the size distribution of cell aggregates during protoplast formation. This made it possible to use changes in optical density (O.D.) to distinguish the three stages in protoplast formation—plasmolysis, maceration and cell wall digestion. During the processes of maceration and cell wall digestion, the O.D. decreased and reached a steady value at the end of each process. Consequently, changes in the O.D. could be used to determine precisely the end of each process. The cell wall digestion process was described by a simple first order reaction model and the rate of protoplast formation (cell wall digestion) was quantitatively evaluated from the rate constant (k) of this reaction. By using the values of k, the optimal enzymatic reaction conditions for isolating protoplasts from C. roseus and N. tabacum cells were determined.     

The influence of Trisenox on actin organization in HL-60 cells

The aim of this study was to show the influence of Trisenox (arsenic trioxide, ATO) on cytoplasmic and nuclear F-actin organization in HL-60 human leukemia cell line. Changes in localization were determined with the use of fluorescence microscopy and flow cytometry. Alterations, in both cytoplasmic and nuclear actin, were observed in cells exposed to ATO. F-actin network underwent accumulation and formed aggregates, that were very often placed under the cell membrane in whole cells and at the periphery of isolated nuclei. Addition of ATO also induced apoptosis and a decrease in G2 phase cells. These results suggest the influence of actin on the formation of apoptotic bodies and also participation of this protein in apoptotic alterations within nuclei, i.e. chromatin reorganization.     

Cell proliferation in ectodermal explants from Xenopus embryos

Summary Dissociated prospective ectoderm cells from Xenopus laevis embryos divide autonomously up to the 17th division cycle of the embryo. To examine the requirements for the further proliferation of these cells, the continuation of cell division in compact ectodermal explants beyond the 17th division cycle has been studied. Such explants develop into aggregates of epidermal cells, as can be shown immunohistochemically with an anti-serum against Xenopus epidermal cytokeratin. Cell division in these explants is comparable to the in vivo proliferation rate at least during the first 24 h of cultivation, that is, well beyond the 17th division cycle. Thus, epidermal cells are provided with all the factors necessary for continued proliferation, but these can be effective only when the cells form tight aggregates. The long-term changes in cell number are complex. Mitotic figures are present until the explants disintegrate after 3–4 days. However, the total cell number per explant does not increase during later development. The production of cells by mitotic divisions is likely to be countered by the loss of cells due to cell death, which is indicated by the presence of pyknotic nuclei.     

The mechanism of nucleation for in vitro collagen fibril formation.

The formation of collagen fibrils from soluble monomers and aggregates by thermal gelation at neutral pH can be divided into two distinct stages: a nucleation phase and a growth phase. Turbidity studies of the kinetics of the precipitation reaction show that the lag-phase time or nucleation reaction time, t′_l, is markedly temperature dependent while the growth reaction time is temperature independent. The activation energy of the nucleation reaction is essentially constant over the temperature range studied. In monitoring the nucleation-phase reaction by various physicochemical techniques, including viscosity, sedimentation equilibrium, and light scattering, no evidence for the formation of aggregates was observed. Enrichment of the initial collagen solution with aggregates accelerates nucleation, but de novo nuclei formation is still required even in highly aggregated collagen preparations. Removal of pepsin and pronase susceptible peptides lengthens the nucleation reaction time and increases the sensitivity of the rate of nuclei formation to changes in ionic strength. Electron microscope studies show the fibrils formed from the protease-treated collagen to be less well organized. With pepsin-treated collagen, subfibrils and obliquely striated fibrils are seen, showing that while microfibrils are formed interactions between them are modulated by the enzyme susceptible peptides in the same way that these regions modulate nuclei assembly. It appears that pepsin and pronase susceptible peptide regions of collagen play a more prominent role in the in vitro assembly of collagen molecules to form D-stagger nuclei and fibrils than do ionic interactions between helical molecular regions. A mechanism of nucleation of collagen fibrillogenesis is discussed.     

Transformation-induced alterations in adhesion : Binding of pre-formed cell aggregates to cell layers

The formation of intercellular adhesions by mouse 3T3 cells and their SV40-transformed derivatives is analysed by measuring the binding of pre-formed aggregates of these cells to cell layers or to a plastic substratum. The rationale for this procedure is to reduce the effects of cell dissociation on quantitative assessments of adhesive interactions. The fibroblasts within the aggregates retain the growth characteristics these cells show in monolayer culture. The proportion of aggregates binding is independent of the number of aggregates added and changes with time in a manner consistent with a first-order process, allowing the percent aggregates binding per unit time to serve as a parameter of intercellular adhesion. The rate of binding in homologous adhesive interactions is slower than in heterologous ones, binding in 3T3SV interactions is slower than in 3T3 interactions, and binding to cellular substrata is slower than to plastic. Binding of 3T3SV aggregates is readily distinguished from binding of 3T3 aggregates by the presence of a brief lag in binding rate, the formation of irregular projections from the bound aggregate, and a differential effect on binding rates of varying the temperature or of treating a single reactant with glutaraldehyde. Thus, there are quantitative and qualitative differences in the adhesive interactions of normal and transformed cells. The distinct binding properties of 3T3SV aggregates and the greater binding rates in heterologous interactions may be relevant to the invasive behavior of transformed cells in vivo.     

Mathematical modeling the kinetics of cell distribution in the process of ligand-receptor binding

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand-receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand-receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.     

Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues.

BACKGROUND: Cellular expression of receptors for the hormones estrogen and progesterone in human mammary tumors is of diagnostic and prognostic value. Ligand binding assays have been replaced by immunohistochemical analysis of receptor expression. However, both of these techniques are slow, and in the ligand-binding assay it is difficult to measure heterogeneity of receptor expression in individual cells. Flow cytometry has been used extensively for monitoring the expression of cellular receptors in hematopoietic tumors but has been of limited value in the analysis of mammary tumors, which are difficult to disaggregate into single cells for flow analysis. Hormone receptors have a predominant nuclear localization, and it is relatively easy to isolate nuclei from paraffin-embedded archival tissues for flow cytometric analysis of receptor expression. METHODS: Thick sections from formalin-fixed paraffin-embedded archival mammary tumors were digested by different enzyme solutions for the isolation of single nuclei. Different fixatives were used to compare the results on subsequent staining of the nuclei for estrogen receptor (ER) expression. Double staining with propidium iodide and fluorescein isothiocyanate labeled secondary antibodies for ER expression was used for multiparametric analysis of ER and DNA content. RESULTS: Digestion of paraffin sections with low concentration of pepsin and detergents was ideal for isolation of single nuclei. Fixation with paraformaldehyde in the presence of Triton X-100 improved staining of the cells. Isolated nuclei had enhanced immunoreactivity compared with the whole cells, and subpopulations differing in reactivity could be identified in the nuclear fractions. Double staining of nuclei for ER expression and DNA content could allow for multiparametric analysis of these two important parameters. CONCLUSIONS: The procedures described can be used for processing of archival paraffin-embedded mammary tumors for monitoring of ER expression and aneuploidy. These two parameters have important diagnostic and prognostic significance in mammary tumors. Laser flow cytometry by providing multiparametric analysis can allow for correlation of these cellular markers with other important cellular and clinical parameters.     

Changes of lysosomal enzymes during hereditary degeneration and histogenesis of retina in mice

Summary In the normal histogenesis of mouse retina localized distribution of acid phosphatase positive granules has been seen around the photoreceptor cell nuclei along the outer limiting membrane. These granules disappear during the development of the rod elements. Temporarily increased activity is also seen along the nuclei of the inner layer adjacent to and in the course of the development of the outer and the inner plexiform layers. Within the inner nuclear layer, the cells at the outer and inner rows develop localized acid phosphatase positive granules which persist in the adult retina. Ganglion cells and the layer of nerve fibres show little change. In the pigment epithelium the enzyme gradually increases. In mice, homozygous for the retinal degeneration gene, degenerating photoreceptor cell nuclei, characterized by perinuclear acid phosphatase staining, can be detected before morphological signs of degeneration. Increased frequency of such nuclei and intensity of staining are recorded with the progress of degeneration. Enzyme activity in the photoreceptor cells, within the inner nuclear layer and in the degenerating photoreceptor cell nuclei is demonstrable using naphthol substrates but not -glycerophosphate. Positive reaction with -glycerophosphate is obtained in these sites in the presence of dimethyl sulphoxide. Existence of differential permeability among the retinal lysosomes is tentatively suggested.     

Intercellular adhesion in butyrate-treated HeLa S3 cultures : Flow cytometric analysis

The application of DNA flow cytometry (FCM) for analysis of sodium butyrate-induced intercellular adhesion in human carcinoma (HeLa S3) cell cultures is described. To prepare cell suspensions for FCM, the monolayers of cells were treated with medium containing 10% serum, 0.2% non-ionic detergent Triton X-100 and 1 μg/ml DNA fluorochrome 4,6′-diamidino-2-phenylindole (DAPI). Total numbers of single cells, and aggregates containing two, three, four or more cells, were determined from DNA histograms. In cultures treated with 5 mM butyrate for 16 h, more than 80% of the cells were aggregated. Intercellular adhesion began to appear 8 h after addition of butyrate, was maximal at 16–24 h and stable in the presence of butyrate, but disappeared 24 h after its removal. Treatment with EDTA (0.2%) dissociated only 50%, whereas trypsin (0.1%) separated all cell aggregates into single cells. Actinomycin D (actD) (0.5 μg/ml) prevented cell adhesion while blocking of cells in S phase with 250 μM 5-fluorouracil or 10 μM methotrexate did not interfere with aggregation. The number of cell aggregates estimated from DNA histograms of butyrate-treated HeLa S3 cultures was the same after staining with DAPI in the presence of Triton X-100 or after vital staining with Hoechst 33342. The DNA content was used as a marker to estimate the cellular composition of aggregates in mixed cultures of HeLa S3 cells and human fibroblasts (U cells). Intercellular adhesion in these cultures was seen only between HeLa S3 cells, indicating specificity of butyrate-induced cell aggregation. FCM provides fast automatic measurement of cell aggregate formation, estimates frequency of aggregates containing different cell numbers, shows participation of cells at different cycle phases in aggregates, and allows the detection of homotypic from heterotypic cell aggregates if the interacting cells have different DNA ploidy.