Über die Vitalfluorochromierung von Mitochondrien in HeLa- un LM-Zellen mit neuen Acridinfarbstoffen

Zusammenfassung Es wird über die Fluorochromierung von Mitochondrien in lebenden Zellen mit neuen Acridinfarbstoffen berichtet. Die verwendeten Fluorochrome sind Derivate von Acridinorange (AO) und von 3-Amino-6-methoxyacridin (AMA) mit verschiedenen Resten in 9- bzw. 10-Stellung (Formelschema 1). Sie sind entweder permanente Farbstoffkationen oder liegen im Kulturmedium als Kationen vor. HeLa-Zellen und Mäusefibroblasten (LM-Zellen) wurden fluorochromiert. Unter günstigen Bedingungen gelang es, die Mitochondrien nicht nur orthochromatisch sondern auch metachromatisch zu färben. Über photodynamische Effekte, die bei der Bestrahlung unter dem Fluoreszenzmikroskop auftreten, wird berichtet. Die Reste in 9- bzw. 10-Stellung begünstigen die Farbstoffakkumulation in den Mitochondrien. vitalfärbung mit den Grundkörpern AO bzw. AMA ergibt demgegenüber metachromatisch gefärbte Lysosomen im orthochromatisch gefärbten Cytoplasma. Der Farbstoff 3-Amino-6-methoxy-9-(2-hydroxyethyl)-acridin fluorochromiert den Kern lebender Zellen.

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The fluorescent staining of mitochondria in living HeLa- and LM-cells with new acridine dyes

Summary The fluorescent staining of mitochondria in living cells with new acridine dyes is reported. The fluorescent dyes used are derivatives of acridine orange (AO) and of 3-amino-6-methoxyacridine (AMA) with various residues in 9- or 10-position (Scheme 1). They are either permanent cationic dyes or cations which are formed by protonation in the culture medium. HeLa cells and mouse fibroblasts (LM cells) have been used for our staining experiments. On favourable conditions we succeeded in staining the mitochondria not only orthochromatically but also metachromatically. Photodynamical effects which have been observed during the exposure of the stained cells in the fluorescence microscope are described. The residues in 9- or 10-position favour the dye accumulation in the mitochondria. Vital staining with the basic compounds AO and AMA however leads to the formation of metachromatically stained lysosomes in the orthochromatically stained cytoplasm. The dye 3-amino-6-methoxy-9-(2-hydroxyethyl)acridine stains the nucleus of living cells.

     

Specific fluorescent bands on chromosomes produced by acridine orange after prestaining with base specific non-fluorescent DNA ligands

Metaphase chromosomes stained with acridine orange exhibit uniform yellow-green fluorescence. Chromosome preparations treated with the non-fluorescent A-T specific antibiotic distamycin A prior to acridine orange staining exhibit longitudinal fluorescent banding patterns similar to those produced by a number of fluorescent R-band techniques. Similarly, chromosome preparations treated with the non-fluorescent G-C specific antibiotic actinomycin D followed by acridine orange staining exhibit Hoechst-type banding patterns. Interactions of various ligand-DNA combinations in solution indicate that the base pair specific antibiotics induce banding patterns by selectively altering acridine orange binding sites in chromosomal regions rich in the particular base pair for which the antibiotic exhibits specificity.     

Automatic cell identification and enrichment in lung cancer. II. Acridine orange for cell sorting of sputum.

Fluorescence spectra were obtained from cells from sputum and pleural effusions stained with different fluorescent dyes and fixed by alternate methods. The spectra were referenced to a standard allowing for fluorescence comparisons of unstained and stained cells under various conditions. The metachromasia of acridine orange-stained cells offers nuclear/cytoplasmic differentiation in a single stain; mithramycin and propidium iodide do not. Unstained cells have an appreciable amount of green (546 nm) fluorescence, as does Carbowax in Saccomanno's preservative. Cytoplasm stained with acidine orange also has appreciable green fluorescence. Consequently, cells with much cytoplasm have high total fluorescence. Cytoplasmic fluorescence is negligible with mithramycin or propidium iodide. The metachromasia of acridine orange-stained cells is altered by alcohol and Carbowax levels in fixatives, keeping other factors constant.     

THE EFFECT OF SONIC OSCILLATION ON THE STRUCTURE AND FUNCTION OF BEEF HEART MITOCHONDRIA

The brilliantly fluorescent cytoplasmic particles that accumulate in HeLa cells treated with acridine orange, previously referred to as acridine orange particles, are shown to represent acid phosphatase positive multivesicular bodies (MVB). Dynamic changes in the ultrastructure of these organelles may be induced by varying the concentration of extracellular dye and the length of exposure to the dye. Low concentrations of dye for long intervals of time lead to marked hypertrophy of the MVB and accumulation of myelin figures within them, the acid phosphatase activity being retained. High concentrations of dye for short time intervals lead initially to a diffuse distribution of dye through out the cytoplasm (cytoplasmic reddening) as viewed in the fluorescence microscope. When cells are stained in this way and incubated in a dye-free medium, the diffusely distributed dye is segregated into MVB within 1 hour. Ultrastructurally, these MVB show dilatation but no myelin figures. The process of dye segregation is energy dependent and will not occur in starved cells. This energy dependence and the occurrence of segregation via dilatation of the MVB rather than ultrastructural transformation, i.e. formation of new binding sites, suggests that the process involves an active transport mechanism. Of the various energy sources supplied to starved cells, only glucose, mannose, and pyruvate are fully effective in supporting dye segregation. Blockage of the tricarboxylic acid cycle with malonate inhibits the effects of pyruvate but not of glucose, demonstrating the efficacy of both the tricarboxylic acid and glycolytic cycles in supplying energy for the process.     

3-Amino-6-methoxy-9-(2-hydroxyethylamino) acridine: A new fluorescent dye to detect mycoplasma contamination in cell cultures

A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations. Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell. In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Acridine Orange—Methyl Green Fluorescent Staining of Nucleoli

The staining of nucleoli with the fluorescent dye acridine orange followed by counter-staining with methyl green differentially stained nucleoli in both plant and animal cells. The nucleoli fluoresced as bright structures highlighted against the quenched fluorescence of the chromatin. This technique provides a simple and highly reproducible method for differential staining of nucleoli.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Acridine orange fluorescence of mast cells as influenced by fixation

Summary The basic fluorochrome dye acridine orange was applied to different tissues of the rat for staining mast cells. This was performed under different fixation conditions in order to assess the effects of fixative fluids on the resulting fluorescent staining. Under the conditions described, acridine orange has constantly revealed mast cells in bright fluorescence in tissues where they are known to be present normally. The results are discussed in reference to each fixative used. But the method failed to show the mucosal mast cells in the stomach and duodenum. This is proposed as a new element for the further characterization of these cells.This work was supported by the Medical Research Council of Canada (Grant No. 2,236).The author is indebted to Mrs. H. Gonzalez for her skillful technical assistance.     

A novel technique for viable cell determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 micrograms/ml; acridine orange, 1-5.0 micrograms/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.     

A Novel Technique for Viable Cell Determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 μg/ml; acridine orange, 1-5.0 μg/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.     

Nuclear death in living Tetrahymena: the case of the haploid nuclei

During sexual conjugation in Tetrahymena the micronucleus divides meiotically, producing four haploid nuclei. While one of these nuclei divides mitotically to yield two genetically identical gametic pronuclei, a stationary pronucleus and a migratory pronucleus, the remaining three haploid nuclei degenerate and disappear. Typically, they migrate to the posterior end of the cell where they remain as residual bodies until they disappear. In the present study we asked whether degenerating haploid nuclei share any properties with apoptotic nuclei. Specifically, we wondered whether they would be stained by "apofluor", a combination of vital fluorescent indicators that differentially stains apoptotic nuclei in living cells. "Apofluor" includes acridine orange, which becomes trapped in acidic compartments and stains lysosomal bodies a brilliant orange-red, and Hoechst 33342, which binds to DNA and stains nuclei bright blue. With this dye combination, while ordinary nuclei stain blue, the apoptotic macronucleus stains first blue-green, then yellow, and finally orange. The progression in color is presumed to be due to the accumulation of protons in the apoptotic nucleus compartment. We found that three of the four post-meiotic haploid nuclei, those that are eliminated, were stained differentially green, then yellow, and then come to be indistinguishable from the orange lysosomal bodies. Differential staining can occur even while the nuclei are located at the anterior ends of the cells, and before the "viable" nucleus divides to form pronuclei. These results indicate that haploid nuclei in the process of degradation are differentially stained in living cells by "apofluor", and that the differential staining occurs early in the elimination process. Further, since the degenerating haploid nuclei are stained by "apofluor" it is likely that they are degraded by a mechanism similar to the elimination of the apoptotic macronucleus.     

Effect of temperature on the plasma membrane and tonoplast ATPases of barley roots : comparison of results obtained with acridine orange and quinacrine

The effect of temperature on the rate of proton transport and ATP hydrolysis by plasma membrane (PM) and tonoplast (TN) ATPases from barley (Hordeum vulgare L. cv CM 72) roots were compared. Rates of proton transport were estimated using the fluorescent amine dyes quinacrine and acridine orange. The ratio between rate of transport and ATP hydrolysis was found to depend on the dye, the temperature, and the type of membrane. The PM ATPase had an estimated Arrhenius energy of activation (Ea) of approximately 18 kilocalories per mole for ATP hydrolysis, and the Ea for proton transport was best estimated with acridine orange, which gave an Ea of 19 kilocalories per mole. The TN ATPase had an Ea for ATP hydrolysis of approximately 10 kilocalories per mole and the Ea for proton transport was best estimated with quinacrine, which gave an Ea of 10 kilocalories per mole. Acridine orange did not give an accurate estimate of Ea for the TN ATPase, nor did quinacrine for the PM ATPase. Reasons for the differences are discussed. Because it was suggested (AJ Pope, RA Leigh [1988] Plant Physiol 86: 1315-1322) that acridine orange interacts with anions to dissipate the pH gradient in TN vesicles, the complex effects of NO₃⁻ on the TN ATPase were also examined using acridine orange and quinacrine and membranes from oats and barley. Fluorescent amine dyes can be used to evaluate the effects of ions, substrates, inhibitors, and temperature on transport but caution is required in using rates of quench to make quantitative estimates of proton fluxes.     

Induced circular dichroism of acridine orange bound to double-stranded RNA and transfer RNA

The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na⁺ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.     

Standardization of the Feulgen-Schiff technique

Summary Four fuchsin analogues (Pararosaniline, Rosaniline, Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches.Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 m.Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.     

Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.     

Fluorescent analysis of irradiation-induced changes in chromatin state

A fluorescent technique with Hoechst-33258 and acridine orange staining was used to assess changes in chromatin state induced by radiation. Fluorochromes with different modes of binding to DNA were chosen. In T lymphocytes chronic irradiation caused a rearrangement of binding between nonhiston proteins and lipids accompanied by conformational changes in DNA, resulting in chromatin condensation. The decrease in fluorescence probably resulted from a reduction in the number of sites accessible for dye binding. After acute irradiation, the fluorescence intensity decreases predominantly due to double-strand breaks.     

The metachromasia of fluorescence of acridine dyes

Summary 1. Fluorescence- and absorption spectra of a number of acridine dyes were measured at several concentrations, in different solvents and at various pH and temperatures.2. In aqueous solutions metachromatic shifts are visible with all dyes — except acridine-yellow — with increasing concentrations, even when the dye is present as di- or trivalent ions in strongly acid solutions.3. Under conditions where reabsorption of the fluorescent light is excluded: completely separated fluorescence and absorption spectra, or measurement of fluorescence in capillaries with 0.8 mm internal bore, metachromatic effects are absent.4. Reasons are given to consider the hypothesis of specific aggregation (formation of dimeric dye-particles) as doubtful. In the case of the acridine dyes the optical properties of the monomeric dye ions are sufficient to explain the metachromatic shifts.With 10 Figures in the Text     

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides.

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.