Mass action and acridine orange staining: static and flow cytofluorometry.

We present results involving an approach to acridine orange staining of intact cells based on basic physicochemical considerations. We show by static microfluorometry of several in vitro and in vivo cell lines that the important parameters for such staining are the molar ratio (Formula: see text), and molar concentration of acridine orange. Differential nuclear DNA and cytoplasmic RNA staining are totally controlled by these two parameters. We show this by a physicochemical model of cell-dye interaction. Finally, we use the method to study the growth parameters of complex in vivo cell populations by automated multiparameter flow microfluorometry. We have explored also, both by static and flow systems, the effect on AO-cell staining of various cell pretreatments such as Triton X-100 and chelating agents.     

Effects of fluorescein isothiocyanate on insulin actions in rat adipocytes.

The effects of fluorescein isothiocyanate II (FITC) on the actions of insulin in rat adipocytes were studied. When adipocytes were incubated with FITC at pH 7.4 (2 mM agent, 8 min), the cells were completely deprived of their specific insulin-binding activity and rendered unresponsive to the hormone. The effect of FITC on the insulin-binding activity was milder at pH 9.0, and cAMP phosphodiesterase in cells exposed to FITC at pH 9.0 was maximally stimulated if the insulin concentration was increased to 100 nM. Under identical conditions, however, glucose transport activity was rendered not only less sensitive but also less responsive to the hormone. When FITC was added to cells after insulin at pH 9.0, the glucose transport activity that had been stimulated by the hormone was considerably reduced. This reduction was largely, but not entirely, prevented if the cells were deprived of ATP, suggesting that FITC (a) elicited the ATP-dependent reversal of the hormonal effect and, simultaneously, (b) mildly inhibited the transport activity per se. Western blot assay of GLUT-4 (a major isoform of glucose transporter in adipocytes) indicated that FITC (a) partially blocked insulin-dependent translocation of GLUT-4 from the intracellular site to the plasma membrane while it (b) induced a mild "insulin-like" effect. It is concluded that FITC at pH 9.0 (a) renders both glucose transport and phosphodiesterase activities less insulin sensitive presumably by modifying the cellular hormone receptor and (b) makes glucose transport activity less responsive to insulin presumably by (i) blocking hormone-dependent translocation of glucose transporter and (ii) mildly inhibiting intrinsic glucose transport activity.     

Analysis of the effect of fluorescence intensity on distributions obtained by flow cytofluorometry

It is shown that one of the major resolution limiting factors in the rapid measurement of fluorescence from individual cells with “fast flow cytofluorometers” is the small number of photons which are counted in each light pulse. A method is described for evaluating this factor for individual systems and for specific cells and stains. Once evaluated, this contribution to the broadening of the distribution can be stripped from the observed distribution to give a closer estimate of the actual distribution of dye in the cell poulation.     

Fluorescein isothiocyanate staining intensity as a probe of hyperthermia-induced changes in chromatin conformation

In a previous report we presented evidence for large increases in fluorescein isothiocyanate (FITC) fluorescent intensity caused by hyperthermia which were not associated with synthesis of heat-shock proteins. We have now refined and considerably extended the measurements of increases in FITC fluorescent intensity caused by hyperthermia within the range 41.0 degrees C to 50.0 degrees C, and associated these with the extent of cell death caused by the hyperthermia. It appears that cell death ensues when the FITC fluorescent intensity has not returned to its baseline value within the time of one cell cycle. If thermotolerance is induced, there is a concomitant reduction in the increase in FITC staining intensity and the extent of cell death. When hyperthermia is followed by acid extraction, an additional increase in FITC staining intensity (above that due to hyperthermia alone) is observed, indicating separate sites of action on basic nuclear proteins. Hyperthermia and acid extraction have related effects on the relationship between FITC and propidium iodide staining. Hyperthermia-induced increases in FITC staining intensity are almost completely reversed by 6.7 mM formaldehyde with a marginal effect on the control FITC staining at this formaldehyde concentration. We suggest that hyperthermia causes extensive dissociation of basic protein-protein binding within nuclear chromatin, and that this may be a contributory cause of hyperthermia-induced cell death.     

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.     

Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures. Some pitfalls in staining and data analysis.

The rapid hypotonic staining procedure developed by Krishan for DNA determinations by flow cytofluorometry has been proven accurate for in vivo cell samples and for cell lines growing in suspension culture. We show that the unmodified procedure may produce distorted DNA histograms when used for staining cells growing in monolayer cultures, however. To eliminate these distortions, it was necessary to avoid the use of trypsin by staining the attached cells directly, using a hypotonic fluorochrome solution to which nonionic detergent was added. Two sublines of HeLa S3 cells are shown to exhibit major differences in their staining characteristics. By using our revised staining procedure, the two sublines appear to produce very satisfactory DNA histograms. However, in only one subline does the S phase fraction calculated from the histograms agree with the autoradiographical labeling index. Mitotic cells remain intact under these staining conditions, and the principal observed effect of nonionic detergents in this case is to decrease the coefficient of variation of fluorescence intensity.     

Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules

The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labeled alpha-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein decreases when the number of fluorescein residues per protein molecule increases; however, after proteolytic digestion the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4 degrees C, 3LL cells bind 750,000 molecules of fluorescein-labeled alpha-glucosylated serum albumin with an apparent binding constant of 2 X 10(6) 1 X mole-1. At 37 degrees C, after 4 hr incubation 2.2 X 10(6) molecules of fluorescent alpha-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.     

Sensitization of surfactants on the chemiluminescence reaction of fluorescein isothiocyanate labeled proteins

The effect of surfactants on fluorescein isothiocyanate (FITC)–bovine serum albumin (BSA)–hypochlorite (ClO⁻), FITC–human serum albumin (HSA)–ClO⁻, FITC–ovoconalbumin (OVA)–ClO⁻, FITC–hemoglobin (Hb)–ClO⁻ systems were investigated with chemiluminescence method by the reversed phase flow injection. It was found that the chemiluminescence (CL) intensity of each system was increased greatly in the presence of cationic surfactants. Cethyltrimethylammonium bromide (CTAB) is the optimal surfactant of these systems. The optimal conditions of the CL reaction and the optimal concentration of CTAB were examined and the function of cationic surfactant CTAB on the CL reaction was also discussed.     

Sensitization of surfactants on the chemiluminescence reaction of fluorescein isothiocyanate labeled proteins

The effect of surfactants on fluorescein isothiocyanate (FITC)-bovine serum albumin (BSA)-hypochlorite (ClO(-)), FITC-human serum albumin (HSA)-ClO(-), FITC-ovoconalbumin (OVA)-ClO(-), FITC-hemoglobin (Hb)-ClO(-) systems were investigated with chemiluminescence method by the reversed phase flow injection. It was found that the chemiluminescence (CL) intensity of each system was increased greatly in the presence of cationic surfactants. Cethyltrimethylammonium bromide (CTAB) is the optimal surfactant of these systems. The optimal conditions of the CL reaction and the optimal concentration of CTAB were examined and the function of cationic surfactant CTAB on the CL reaction was also discussed.     

Fluorescence and viability of Proteus mirabilis stained directly with fluorescein isothiocyanate

Grunberg, E. (Hoffman-La Roche, Inc., Nutley, N.J.), and R. Cleeland. Fluorescence and viability of Proteus mirabilis stained directly with fluorescein isothiocyanate. J. Bacteriol. 92:23-27. 1966.-Washed cell suspensions of Proteus mirabilis, under the proper conditions, stained well with fluorescein isothiocyanate with little or no loss of cell viability. The speed and intensity of the reaction was dependent on both the concentration of dye and pH. Within a range of pH 3.0 to 10.0, staining was most rapid at pH 5.0 to 6.0, with a slower and less intense reaction occurring at the other pH values. As the concentration of dye at either pH 5.0 or 9.0 was increased from 10 to 1,000 mug/ml, there was an increase in the rate of staining but a decrease in cell viability. After 24 hr of incubation at 4 C, pH 5.0, and a dye concentration of 10 mug/ml, all cells were stained, the majority exhibiting intense fluorescence with little or no loss of viability noted. In preliminary experiments with Staphylococcus aureus, similar results were obtained. Of various other fluorescent dyes tested, only rhodamine isothiocyanate was found to give satisfactory staining.     

Effects of hyperthermia on microtubule organization and cytolytic activity of murine cytotoxic T lymphocytes

When murine cytotoxic T lymphocytes (CTL) are heated at 42 degrees C for 30 min their ability to lyse their target cells (TC) is severely impaired. When the CTL are allowed to recover at 37 degrees C, a partial recovery of cytolytic activity that peaks within 6 h is observed. A dye exclusion assay demonstrated that such a heat shock does not affect the viability of the CTL and direct microscopic observations established that their ability to bind to TC is not impaired. Therefore, the step or steps inhibited by hyperthermia are subsequent to TC recognition and binding. Kupfer et al. ((1983) Proc. Natl. Acad. Sci. USA 80, 7224-7228) demonstrated that upon binding to an appropriate TC, a rapid orientation of the Golgi apparatus and the microtubule organizing center (MTOC) occurred within the CTL so that the two organelles face the TC. This orientation is a prerequisite for efficient TC lysis. We have shown by immunofluorescence and confocal microscopy, using a monoclonal antibody to tubulin and a rabbit autoimmune serum that binds a centriole-associated protein, that the organization of the MTOC-microtubule array is disrupted by hyperthermia. EM suggests that this disorganization of the microtubules may result from an aggregation of the pericentriolar material. The recovery of cytolytic activity is coincident with the reorganization of the microtubules about the MTOC. These findings suggest that the initial inhibitory effect of hyperthermia on CTL function results from the disruption of microtubule organization.     

The reaction of fluorescein isothiocyanate with thiols: a method for assay of isothiocyanates

The reversible formation of dithiourethanes from fluorescein isothiocyanate and mercaptoethanol or mercaptoethylamine has been studied. It is shown that it is possible to use the reaction as the basis for a spectrophotometric titration of a number of isothiocyanates with mercaptoethanol.     

The application of fluorescein isothiocyanate and high-performance liquid chromatography for the microsequencing of proteins and peptides

Azocoll, an insoluble, ground collagen to which a bright-red azodye is attached has been widely used for the assay of proteolytic enzymes. Earlier studies showed that hydrolysis of azocoll progressed linearly as a function of proteinase concentration but in an exponentially increasing manner as a function of time. No explanation for the latter behavior has been offered. We have found that assays of both crude extracts of Bacillus subtilis and commercial preparations of subtilisin BPN' gave linear rates of hydrolysis of azocoll as a function of protease concentration; however, both gave increasing rates of hydrolysis of azocoll as a function of time. In attempting to improve and standardize proteolytic assays using azocoll we have found: (a) the absorption maximum of solubilized azocoll at pH 7.8 is 516 nm and is not significantly altered at acid pH; (b) assays which are perfectly linear as a function of time can be obtained by using azocoll that has been vigorously prewashed with buffer; (c) the soluble filtrate removed by prewashing can regenerate the nonlinear time courses previously observed; and (d) the rate of hydrolysis of azocoll can be varied by a factor of 3 by varying the rates of agitation of the assay tubes. In summary, to obtain reproducible, linear assays it was essential to prewash commercial azocoll and agitate reaction tubes vigorously.     

Screening for supra-additive effects of cytotoxic drugs and gamma irradiation in an in vitro model for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. A wide variety of treatment modalities is available for palliative therapy of HCC, although there is no strong evidence that these treatments can have a significant impact on survival. The aim of this work was to screen cytotoxic drugs relevant in the treatment of HCC for enhancement of the effect of irradiation in an in vitro model. As the majority of patients presenting with HCC suffer reduced liver function, attention was paid to low-dose effects of the cytotoxic drugs tested. To reflect this situation in vivo, multicellular tumor aggregates or "spheroids" of HepG2 cells were cultured and exposed to gamma irradiation alone or in combination with cisplatin for 4 h, gemcitabin for 4 or 24 h, or 5-fluorouracil for 4 h. In one experiment, the spheroids were cultured for 4 weeks in multiwell plates that allowed adhesion. Measurement of two-dimensional spheroid outgrowth was made every week for each spheroid. This kind of growth depends on the proliferation and motility of the cells that form the spheroid. In a second experiment, toxicity was evaluated by comparative growth curves by means of a three-dimensional growth assay and by histology. Supra-additive effects lasting for 4 weeks were observed for all drugs tested in combination with a gamma irradiation of 10 Gy.     

A conjugate of cellulase with fluorescein isothiocyanate: a specific stain for cellulose.

A fluorescent technique has been developed for in situ staining of cellulose. The staining agent in conjugate of cellulase and fluorescein isothiocyanate (FITC). Application of this agent does not disturb intercellular or intracellular substances. The technique depends on the specific binding of the fluorescent labeled enzyme to its substrate. The stain has been tested on cell-free noncellulose polysaccharides similar to cellulose and does not stain them. The technique has been used to localize cellulose during the life cycle of Dictyostelium discoideum with results that correspond to previous work using other methods.     

A method for detecting proteins immobilized on nitrocellulose membranes by in situ derivatization with fluorescein isothiocyanate

A method for the fluorescent staining of proteins on nitrocellulose filters is described. The single step procedure uses a 100 microgram/ml solution of fluorescein isothiocyanate in sodium carbonate buffer, pH 9.5. The proteins are visible under uv light within 30 s and the staining reaction is virtually complete after 10 min. The method can detect a minimum of 50 ng protein/band providing a sensitivity similar to that obtained with anionic dye stains. The method is suitable for blots prepared from both isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. The fluorescently labeled proteins can be probed using immunochemical techniques with the retention of fluorescence. The method can therefore be used to accurately locate antigens among a number of proteins and allows the sensitive and rapid detection of marker proteins directly on the blot.