An improved method to investigate staining kinetics in single cells

Summary 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.     

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.     

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.     

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.     

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.     

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.     

Co-migration and internalization of transferrin and its receptor on K562 cells

The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC- transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fab-antitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4 degrees C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20 degrees C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20 degrees C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.     

Proliferation-associated expression of DNA methyltransferase in human embryonic lung cells

The cell cycle-dependent and proliferation-associated expression of the enzyme DNA methyltransferase has been evaluated immunocytochemically in synchronized L-132 human embryonic lung cells, using the anti-DNA methyltransferase monoclonal antibody M1F6D7/5C10. DNA methyltransferase-reactivity was firstly seen in mid-G1 cells. An intense and granular reaction in the cell nuclei with a sparing of the nucleoli was observed in addition to a homogenous and faint cytoplasmic staining. The staining intensity in the cell nuclei increased progressively up to mitosis. In early mitotic cells an intense perichromosomal staining was observed in addition to a homogenous staining of cyto- and karyoplasm after the resolving of the core membrane. In late mitosis the staining intensity decreased rapidly. Early G1 cells and density inhibited, resting G0 cells showed no DNA methyltransferase reactivity at all. Our results indicate that anti-DNA methyltransferase monoclonal antibodies could become valuable tools to detect proliferating cells in cell cultures and tissues.     

Flow cytometric cell division tracking using nuclei

BACKGROUND: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions. METHODS: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. RESULTS: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined. CONCLUSIONS: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle.     

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.     

Chromatin condensation in hamster sperm: A flow cytometric investigation

In this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine. © 1994 Wiley-Liss, Inc.     

The forward rate of binding of surface-tethered reactants: effect of relative motion between two surfaces

The reaction of molecules confined to two dimensions is of interest in cell adhesion, specifically for the reaction between cell surface receptors and substrate-bound ligand. We have developed a model to describe the overall rate of reaction of species that are bound to surfaces under relative motion, such that the Peclet number is order one or greater. The encounter rate between reactive species is calculated from solution of the two-dimensional convection-diffusion equation. The probability that each encounter will lead to binding depends on the intrinsic rate of reaction and the encounter duration. The encounter duration is obtained from the theory of first passage times. We find that the binding rate increases with relative velocity between the two surfaces, then reaches a plateau. This plateau indicates that the increase in the encounter rate is counterbalanced by the decrease in the encounter duration as the relative velocity increases. The binding rate is fully described by two dimensionless parameters, the Peclet number and the Damk?hler number. We use this model to explain data from the cell adhesion literature by incorporating these rate laws into "adhesive dynamics" simulations to model the binding of a cell to a surface under flow. Leukocytes are known to display a "shear threshold effect" when binding selectin-coated surfaces under shear flow, defined as an increase in bind rate with shear; this effect, as calculated here, is due to an increase in collisions between receptor and ligand with increasing shear. The model can be used to explain other published data on the effect of wall shear rate on the binding of cells to surfaces, specifically the mild decrease in binding within a fixed area with increasing shear rate.     

Mesothelioma Symposium 11.30–12.30 Tuesday 16 September 2003. Cytopathology of malignant mesothelioma

The diagnosis of malignant mesothelioma on the cytology of serous effusions is a two‐phase process. First is to determine that the effusion is malignant based on morphological features such as a highly cellular fluid with many large three dimensional cell aggregates, and/or the recognition of minor malignant criteria including prominent cell engulfment, uniformly present very prominent nucleoli, or the finding of very large (giant) cells. In cell block sections, strong positive staining with EMA often with cell membrane accentuation provides compelling support for a cytological diagnosis of malignancy. Second is to recognize that the malignant cells have a mesothelial phenotype and do not represent metastatic malignancy (usually adenocarcinoma). Criteria in support of mesothelioma include the lack of a ‘two cell’ population, that is one native (mesothelial) and one foreign (metastatic), cells with abundant dense staining cytoplasm, the presence of ‘windows’ where mesothelioma cells lie in close apposition and intracytoplasmic glycogen presenting either as small peripheral vacuoles on MGG stained smears or large yellow refractile crescents on Papanicolaou stained smears. In addition, mesothliomas often possess connective tissue stromal cores occurring as either well‐formed collagen within papillary aggregates or lying free as pink (MGG) or light green (Pap) amorphous material in the background of the smear or in loose association with mesothelioma cells. Finally small orange staining squamous‐like cells can occasionally be identified and sometimes this may be a very prominent finding and has resulted in the false impression of a squamous cell carcinoma. Almost certainly these cells represent apoptotic tumour cells. The connective tissue mucin hyaluronic acid may be found as a net‐like pattern in the smear background or as large hard‐edged magenta‐stained vacuoles on MGG‐stained smears. Cell block sections provide architectural information and it is usually possible to separate mesothelioma aggregates with their cuboidal cells, central nuclei and abundant dense cytoplasm arranged in solid, papillary or hollow clusters from those of adenocarcinoma with less dense, often foamy cytoplasm, often composed of columnar cells with elongated nuclei. Aggregate form in adenocarcinoma can be variable but true acini are a rare finding. These cell block sections provide an ideal medium for histochemistry (PAS with and without diastase digestion) and immunocytochemistry. By using a panel of antibodies (Calretinin and CK 5/6, BerEp4, CEA, B72.3) it is almost always possible to distinguish mesothelioma from metastatic adenocarcinoma. Calretinin and CK 5/6 positive staining and absent staining with BerEp4, CEA and B72.3 is considered diagnostic of mesothelioma.     

Use of preformed cell aggregates and layers to measure tissue-specific differences in intercellular adhesion

The binding of aggregates formed from various 7-day chick embryo tissues to cultured cell layers was analyzed 24 hr following trypsin dissociation of the tissues. The proprotion of aggregates binding is independent of the number of aggregates added, and changes with time over 60 min in a manner consistent with a first-order process. The adhesive parameter measured, the percentage of aggregates binding to cell layers per unit time, varies slightly with aggregate size but is not dependent upon the probability of collision of the aggregate with the layer. The rate of binding and the effect of modifiers of binding (temperature, inhibitors of oxidative metabolism and glutaraldehyde) are substantially different for neural retina interactions than for liver or heart interactions, suggesting that retina cells may form intercellular bonds via a mechanism distinct from that of liver or heart cells. The rate of binding between like tissue types is, with one exception, greater than between unlike types. Glutaraldehyde treatment of only one of the reactants abolishes this adhesive specificity. Aggregate binding provides a means of quantitatively assessing intercellular adhesion which has the advantage of reducing the effects of trypsinization on measurements of adhesion, and therefore lends itself to the investigation of cellular consequences of adhesion.     

A method for staining 3T3 cell nuclei with propidium iodide in hypotonic solution

A modification of the propidium-iodide hypotonic sodium citrate method has been developed specifically for high-resolution staining of mouse 3T3 cell nuclei for analysis by flow cytometry. The method employs a brief treatment of cells at 37 degrees C with Triton X-100 and RNAse in the presence of propidium iodide in hypotonic sodium citrate, followed by restoration to isotonicity with NaCl. The average CV obtained for the G1 peak was 3.5%, and the samples were stable for 1-2 weeks at 4 degrees C. Compared to this technique, previously described propidium iodide-staining methods gave poor resolution with 3T3 cells.     

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.