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.     

Ligands with affinity to specific sequence of DNA base pairs. XII. The synthesis and cytological studies of dimeric Hoechst 33258 molecules

We synthesized dimeric Hoechst dye molecules composed of two moieties of the Hoechst 33258 fluorescent dye phenolic hydroxy groups of which were tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human premonocytic leukemia HL60 cells was observed for all the three fluorescent dyes. The most contrast pattern was obtained for the bis-Hoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4',6-diamidino-2-phenylindole. The ability to penetrate into the live human fibroblasts was studied for the three bis-Hoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bis-Hoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bis-Hoechst was considerably weaker than that of the fixed cells. The bis-Hoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.     

The significance of azo-reduction in the mutagenesis and carcinogenesis of azo dyes

Azo dyes are widely used in textile, printing, cosmetic, drug and food-processing industries. They are also used extensively in laboratories as either biological stains or pH indicators. The extent of such use is related to the degree of industrialization. Since intestinal cancer is more common in highly industrialized countries, a possible connection may exist between the increase in the number of cancer cases and the use of azo dyes. Azo dyes can be reduced to aromatic amines by the intestinal microflora. The mutagenicity of a number of azo dyes is reviewed in this paper. They include Trypan Blue, Ponceau 3R, Pinceau 2R, Methyl Red, Methyl Yellow, Methyl Orange, Lithol Red, Orange I, Orange II, 4-Phenylazo-Naphthylamine, Sudan I, Sudan IV, Acid Alizarin Violet N, Fast Garnet GBC, Allura Red, Ponceau SX, Sunset Yellow, Tartrazine, Citrus Red No. 2, Orange B, Yellow AB, Carmoisine, Mercury Orange, Ponceau S, Versatint Blue, Phenylazophenol, Evan's Blue and their degraded aromatic amines. The significance of azo reduction in the mutagenesis and carcinogenesis of azo dyes is discussed.     

Metachromatic effects and binding of organic cations to energized submitochondrial particles

Energization of submitochondrial particles results in a marked increase of binding, measured as number of sites and binding constants, of the cationic dyes Acridine Orange and Neutral Red. The binding of the dyes is accompanied by spectral changes which are identified as metachromatic effects. The findings are interpreted in terms of interaction with electron-negative sites and stacking of the dye molecules.     

Alterations induced in mouse chromosomes by restriction endonucleases

Fixed chromosomes of mouse have been treated with Alu I, Eco RII, Hind III or Bam HI restriction endonucleases and subsequently stained with either Giemsa, Ethidium Bromide or Acridine Orange. The results obtained have been discussed in the light of preferential or non-preferential extraction of DNA from specific chromosome areas following enzyme digestion. The possible involvement of a particular structural organization of some classes of heterochromatin has been hypothesized to account for the findings after Alu I or Eco RII treatment. The meaning of the Giemsa banding observed after Hind III or Bam HI digestion has also been considered, in comparison to the different stain responses obtained by using a DNA-specific dye such as Ethidium Bromide.     

DNA Sequence-Specific Ligands: XII. Synthesis and Cytological Studies of Dimeric Hoechst 33258 Molecules

We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 fluorescent dye with the phenolic hydroxy groups tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human HL60 premonocytic leukemia cells was observed for all the three fluorescent dyes. The most contrasting pattern was obtained for the bisHoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4′,6-diamidino-2-phenylindole. The ability to penetrate into live human fibroblasts was studied for the three bisHoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bisHoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bisHoechst was considerably weaker than that of the fixed cells. The bisHoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 385–393.Original Russian Text Copyright © 2005 by Gromyko, Popov, Mosoleva, Streltsov, Grokhovsky, Oleinikov, Zhuze.     

Cyanine dyes for the detection of double stranded DNA

Twenty three novel cyanine dyes have been applied as fluorescent stains for the detection of nucleic acids in agarose gel electrophoresis. Significant fluorescence enhancement of these dyes in the presence of double stranded DNA was observed. Five dyes offered superior sensitivity in the detection and quantification of DNA, over Ethidium Bromide, the most commonly used nucleic acid stain.     

An application of Acridine Orange fluorescent staining to the micronucleus test

Acridine Orange fluorescent staining was applied to the micronucleus test in mice and rats. Micronuclei emitted bright green fluorescence and were easily distinguished from micronucleus-like inclusions or contaminants. In rat bone-marrow cells, micronuclei with green fluorescence could be easily distinguished from granules accidentally dispersed from broken mast cells, which showed bright red fluorescence. Therefore, it is recommended that the Acridine Orange staining method be used to provide more reliable data in the micronucleus test.     

Optimization of an Acridine Orange–Bisbenzimide Procedure for the Detection of Apoptosis-Associated Fluorescence Colour Changes in Etoposide-Treated Cell Cultures

This study was initiated in order to investigate the possibility of improving fluorescence microscopy as a method for evaluating apoptosis in cells by combining two fluorescent dyes with different staining characteristics. Cells were vitally stained with bisbenzimide (1.3 microM) and Acridine Orange (6.6 microM) and observed using the following filter configuration: excitation 380 nm, beamsplitter 395 nm and longpass filter 397 nm. Control cells exhibited clear blue fluorescent nuclei and red fluorescing lysosomes. In cells treated with etoposide to induce apoptosis, two distinct occurrences were observed: a change in the spectrum of emitted light from bisbenzimide bound to the nuclear region and an increase in lysosomal Acridine Orange fluorescence. The two occurrences together permit a more unbiased detection of apoptosis than most assays. Only one filter set is required for evaluation and the resulting images can be easily evaluated visually or processed further by image analysis.     

Sensitized fluorescence in polynucleotide-dye complexes and the problem of energy transfer in polynucleotides

New results on sensitization of fluorescence of Profiuvine, Acridine Orange and Ethidium Bromide complexes wilh double stranded poly A-poly U upon ultraviolet irradiation are presented. Together with older results on dye DNA complex, they are interpreted to show that base to base transfer plays essentially no role compared to direct base to dye transfer. A lower limit of base to base transfer time (which is at the same time a higher limit for life time of fluorescence) in DNA (2. 10^?11 sec.) and poly A-poly U (2. 10^?13 sec.) is obtained which cast some doubt upon the validity of calculations of optical properties using the exciton (strong coupling) approximation.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Summary Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-U.V irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.In honour of Prof. P. van Duijn     

Behavior of theophylline-sensitive T lymphocytes in viral A, B, non-A, non-B hepatitis. I. Methodological aspects

To evaluate the behaviour of the Theophylline-sensitive T lymphocytes subpopulation some modifications of the standard procedure are proposed. Lymphoprep purified lymphocytes were counted in a Neubauer hemocytometer after Acridine Orange stain, viability was evaluated by Ethidium Bromide counterstain and monocytes contamination was evaluated by the peroxidase stain. Sheep red blood cells were treated with AET, Theophylline was used at 3 mM (final concentration) and the results compared with untreated lymphocytes; the enumeration of the rosetting lymphocytes was facilitated by adding Acridine Orange prior to the resuspension. The modifications described were able to increase the % of rosetting T lymphocytes, to eliminate differences depending by different lots of sheep red blood cells and to decrease differences depending by subjective evaluation of the rosetting T lymphocytes.     

Two different types of apoptosis in thymocytes

Thymocyte cell death was investigated after UVA-irradiation (365 nm) of the primary thymocyte culture in vitro. To determine the mode of cell death two fluorescent DNA-binding dyes (Acridine Orange and Ethidium Bromide) were used along with flow cytometry. Thymocytes undergo apoptosis spontaneously, but a percentage of apoptotic cells was seen to increase in a dose dependent manner after thymocytes exposition to 1-100 J/m2 UVA. These doses are lower than those commonly used for apoptosis induction in other lymphoid cells. Using flow cytometry, we demonstrated that UVA-irradiation induced two different types of apoptosis. The one referred to as the "fast" apoptosis was recorded within 1-4 h following irradiation, whereas the other one, called the "delayed" apoptosis occurred within 3-24 h after irradiation. After UVA-irradiation, the activation of the former prevented the development of the latter; whereas the inhibition of delayed apoptosis brought about the induction of the fast apoptosis in thymocytes. The interrelation between the fast and delayed types of apoptosis in thymocytes can be modulated by cycloheximide, an inhibitor of protein synthesis.     

A Rapid Viability Test for Sclerotia with Fluorescein Diacetate

Four different fluorescent dyes Acridine Orange (AO), Fluorescein Diacetate (FDA), Calcofluor White M2R (CW), and Europium (III) Thenoyltrifluoroacetonate, 3-Hydrate (Eu(TTA)₃) were tested to distinguish rapidly between dead and living sclerotia from Sclerotinia trifoliorum. FDA, 0.01% diluted in phosphate buffer, had the shortest staining period and was easy to handle. It is especially suitable to test large numbers of sclerotia for viability. Furthermore the preparation of the samples for the staining procedure is described in this paper.     

DNA interaction, cytotoxicity, and radiosensitization with PtCl4(Nile Blue)2 and PtCl4(Neutral Red)2

Complexes of the positively charged, nuclear staining, quinone-imine dyes Nile Blue and Neutral Red with negatively charged tetrachloroplatinum (II) have been prepared in an effort to form neutral drugs which could gain ready access to the cellular nucleus and deliver significant quantities of the reactive tetrachloroplatinum anion to the vicinity of the DNA. Elemental analysis showed that both the Nile Blue and Neutral Red complexes with tetrachloroplatinum (II) comprised 2 mol of dye and 1 mol of tetrachloroplatinum, forming Pt(Nile Blue)2 and Pt(Neutral Red)2. Exposure of superhelical pBR322 DNA to the complexes or the dyes for 24 h followed by agarose gel electrophoresis showed that Neutral Red and Pt(Neutral Red)2 had little effect on DNA conformation, but that both Nile Blue and Pt(Nile Blue)2 could produce single-strand DNA breaks in a dose-dependent fashion. Studies in exponentially growing asynchronous, hypoxic, and normally oxygenated EMT6 cells at normal pH (7.40) and pH 6.45 demonstrated that neither dye was highly toxic, but that both complexes were capable of producing significant cytotoxicity. Both complexes killed normally oxygenated cells more efficiently than hypoxic cells, but Pt(Neutral Red)2 was more cytotoxic at pH 6.45, while Pt(Nile Blue)2 killed significantly more cells at normal pH. Both complexes decreased the survival of hypoxic EMT6 cells as indicated by the slope of the radiation survival curve [dose modifying factor (DMF) 2.90 for Pt(Nile Blue)2 and 1.45 for Pt(Neutral Red)2]. Studies with the FSaIIC murine tumor showed that both complexes were active radiosensitizing agents in vivo [DMF 1.76 for Pt(Nile Blue)2 and 1.25 for Pt(Neutral Red)2]. These results indicate that these new platinum complexes have characteristics which may make them and similar complexes effective radiosensitizing agents in humans.     

Effects of counterstaining with DNA binding drugs on fluorescent banding patterns of human and mammalian chromosomes

Pairs of fluorescent A-T specific dyes and nonfluorescent agents with similar or complementary base pair binding specificity were used to analyse the extent to which banding patterns in human chromosomes obtained by fluorescent staining can be modified by counterstaining. By testing a variety of different combinations of drugs, essentially three types of alterations were observed. Enhanced contrast of specific heterochromatic regions was obtained with pentamidine, or netropsin, in conjunction with the fluorescent stains Hoechst 33258, DAPI or DIPI, the resulting banding patterns being similar to that reported for distamycin A plus DAPI (DA-DAPI banding [21]. Uniform quenching of Hoechst 33258, DAPI or DIPI fluorescence was induced by counterstaining with stilbamidine or berenil. The combination of echinomycin with DAPI resulted in an improved contrast of DAPI banding on chromosome arms and pale fluorescence on major autosomal C band regions. In addition, a subdivision of the heterochromatic part of the Y chromosome may be discerned by this latter technique.     

<Emphasis Type="Italic">In Vitro</Emphasis> Characterization and Evaluation of the Cytotoxicity Effects of Nisin and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines

The aim of this study was an in vitro evaluation and comparison of the cytotoxic effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical properties, including formation, size, and shape, were studied using hemolysis, fluorescein isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading efficiency and release kinetics were examined to assess the potential impact of this formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays. Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange (AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter nanoparticles was about 85–90%. The hemolysis test represented their non-cytotoxicities and the FITC images indicated their entrance inside the cells. An increase in the percentage of apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.     

An investigation of capacitation and the acrosome reaction in dog spermatozoa using a dual fluorescent staining technique

The processes of capacitation and acrosomal exocytosis of dog spermatozoa in vitro have yet to be fully investigated. Firstly, we investigated the effectiveness of a technique for staining dog spermatozoa with the fluorescent labels Hoechst 33258 and chlortetracycline. A modified fluorescence microscopy staining method was shown to be effective for the assessment of both viability and functional status in this species. Secondly, the presence of the ionophore A23187 in culture medium was shown to promote capacitation and the acrosome reaction of dog spermatozoa. We have therefore established that this dual fluorescent staining method can be used for monitoring these events in the dog, and it may be useful in future studies of optimal in vitro culture conditions.