Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.     

Fluorochrome staining of multilamellar liposomes.

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 x magnification). The fluorochrome is seen to be accumulated in the liposomal membranes. Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time. Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

A critical evaluation of the specificity of aniline blue induced fluorescence

Summary Commercial aniline blue dyes are heterogeneous and variable. We have isolated and purified the fluorochrome from water soluble aniline blue. This fluorochrome fluoresces weakly with a maximum emission around 455 nm but the fluorescence is shifted to longer wavelengths (500–506 nm) when complexed with isolated -1,3-glucans, cellulose or mixed-linked glucans. A similar intense fluorescence is observed in sieve plates, new cell walls and around pitfields in the presence of the fluorochrome. The fluorescence induced by the aniline blue fluorochrome does not specifically indicate the presence of -1,3-glucans. Indeed most wall features are induced to fluoresce to some extent by the fluorochrome. However, fluorescence is modified by lignins and phenolics. Furthermore the intense fluorescence induced in the traditional callose sites, sieve plates, around pitfields and in new cell walls is probably related to localized differences in the structural packing of wall polymers.     

Fluorochrome Staining of Multilamellar Liposomes

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 × magnification). The fluorochrome is seen to be accumulated in the liposomal membranes.

Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time.

Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

A dual fluorochrome probe for imaging proteases

Near-infrared fluorescence (NIRF) optical probes have been able to provide a noninvasive assessment of enzyme activity for a number of different enzymes and types of pathology. Here we describe a dual fluorochrome enzyme-activatable probe featuring one NIRF fluorochrome that is activated by protease activity and a second fluorochrome that is protease resistant and serves as an internal standard. The probe was prepared by attaching Cy7 directly to an amino-CLIO, an amine functional cross-linked iron oxide (CLIO) nanoparticle carrier, in a protease resistant manner. Cy5.5 was attached to a protease sensitive polyarginine peptide spacer, also attached to amino-CLIO. In vitro and in vivo the ratio of the Cy5.5 to Cy7 fluorescence was increased by protease, reflecting the increase in Cy5.5 fluorescence by protease in the vicinity of the probe. In vitro and in vivo the absolute values of the Cy5.5 and Cy7 fluorescence reflected lesion size and the distance of lesions from the surface, while the ratio of Cy5.5 to Cy7 fluorescence obtained was constant and independent of lesion size and depth. The dual fluorochrome probe, and related dual wavelength imaging method, represents a novel approach for imaging protease activity in vivo.     

Clearly differentiated and stable chromosome bands produced by a spermine bis-acridine, a bifunctional intercalating analogue of quinacrine

Characteristic fluorescent banding patterns on human metaphase chromosomes are produced by treating chromosome preparations directly with a spermine bis-acridine fluorochrome (CMA)₂S. The clearly differentiated bands are similar to those produced by quinacrine (Q-banding), but show enhanced definition between bright and dull regions as compared with the banding patterns obtained by the quinacrine technique. In addition, the bands on chromosomes produced by (CMA)₂S show insignificant fluorescence fading over extended periods of excitation. Solution interactions between DNA and (CMA)₂S showed a greater fluorescence differential between fluorescence enhancement by the alternating polymers poly d(A-T) · poly d(A-T) and fluorescence quenching by the polynucleotide poly d(G-C) · poly d(G-C) for this fluorochrome than was observed for quinacrine. The increased definition in Q-type bands produced by the spermine bis-intercalating derivative and the lack of fluorescence fading make this fluorochrome an excellent one for routine clinical cytogenetic analysis.     

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome. The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.     

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

Summary We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome.The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.     

An error in interpretation of double-reciprocal plots and Scatchard plots in studies of binding of fluorescent probes to proteins,and alternative proposals for determining binding parameters

One of the objects of experiments in which a fluorochrome is added to suspensions of cell membranes is to determine the parameters n and K _D , the capacity of unit mass of protein to bind fluorochrome and the dissociation constant, respectively. Currently, these are estimated from Scatchard plots, construction of which first requires that observed fluorescence intensity be converted to moles of bound fluorochrome. This in turn is said to be possible by analysis of the intercept of a plot of reciprocal fluorescence intensity against reciprocal protein concentration. However, analysis of the classical mass action equilibrium equation, upon which the foregoing procedures are said to be based, reveals that the intercept of the double-reciprocal plot always underestimates the desired value. The error is formalized and shown to increase without bound with fluorochrome concentration. The error in turn leads to erroneous assessment of n and K _D . Alternative methods for calculating the desired parameters are proposed, based on direct plots of fluorescence intensity.     

2-hydroxystilbamidine isethionate: a new fluorochrome for use in general pathology. I. The selective staining of DNA, mucosubstances and elastic fibres

2 mg of 2-Hydroxystilbamidine isethionate when dissolved in 50 ml 0.1 M citric acid produced nuclear fluorescence in paraffin sections. Pre-hydrolysis in 5N HCl at room temperature increased selectivity of nuclear fluorescence. The addition of 100-200 mg sodium metabisulphite to the fluorochrome solution and preoxidation in periodic acid produced selective fluorescence of mucosubstances. Pre-oxidation with potassium permanganate induced selective fluorescence of elastic fibres. Yellow nuclear fluorescence contrasted clearly with blue/white fluorescence of mucosubstances and elastic fibres when excited with UV light. Unwanted nuclear fluorescence was quenched with 5% iron alum solution. Mast cells selectively fluoresced in acid alcoholic solutions of the fluorochrome. The procedures described were simple and rapid and produced permanent fluorescent preparations. The metachromatic fluorescence of nuclei in contrast to that of mucosubstances and elastic fibres eliminated the need for counterstaining.     

植物小孢子母细胞减数分裂过程中胼胝质染色的新方法

利用改良苯酚品红-苯胺蓝压片法,观察小孢子母细胞减数分裂过程中胼胝质的动态变化。使用该方法简便、快速且省时,获得的照片颜色鲜艳,细胞质呈红色,染色体为深红色,胼胝质呈黄绿色荧光,对比明显,有三维效果。单用改良苯酚品红染液对新鲜材料进行压片,在蓝光激发下,细胞质与染色体呈红色荧光,染色体清晰。实验结果表明,改良苯酚品红染液可作为荧光染料代替DAPI及H33258等昂贵的核染料,从而降低实验成本。     

2-Hydroxystilbamidine isethionate: A new fluorochrome for use in general pathology

Summary 2 mg of 2-Hydroxystilbamidine isethionate when dissolved in 50 ml 0.1 M citric acid produced nuclear fluorescence in paraffin sections. Pre-hydrolysis in 5N HCl at room temperature increased selectivity of nuclear fluorescence. The addition of 100–200 mg sodium metabisulphite to the fluorochrome solution and preoxidation in periodic acid produced selective fluorescence of mucosubstances. Pre-oxidation with potassium permanganate induced selective fluorescence of elastic fibres. Yellow nuclear fluorescence contrasted clearly with blue/white fluorescence of mucosubstances and elastic fibres when excited with UV light. Unwanted nuclear fluorescence was quenched with 5% iron alum solution. Mast cells selectively fluoresced in acid alcoholic solutions of the fluorochrome. The procedures described were simple and rapid and produced permanent fluorescent preparations. The metachromatic fluorescence of nuclei in contrast to that of mucosubstances and elastic fibres eliminated the need for counterstaining.     

A rapid method for accurate DNA measurements in single cells in situ using a simple microfluorimeter and Hoechst 33258 as a quantitative fluorochrome

A method is described for the rapid, accurate measurement of single cells in situ using microfluorimetry. This method involves a shutter system, which allows irradiation of single cells for fractions of a second and a peak fluorescence intensity recording device. In this way errors due to fluorochrome fading are almost eliminated and standard deviations of less than 5% are obtained. Hoechst 33258 has been used as a quantitative fluorochrome. Optimum fixation and staining conditions on glass and plastic tissue culture vessels are described.     

A simple fluorescence technique to stain the plasma membrane of human neutrophils

Three different fluorochromes were tested for their ability to label the plasma membrane proteins of neutrophils without labelling intracellular structures. A fluorescence quenching technique was used to differentiate between extra- and intracellularly localized fluorescence. Fluorescamin and fluoresceinisothiocyanate were shown to stain intracellular structures as well as the plasma membranes of the cells. Another fluorochrome, Evans Blue, is proposed since this dye was shown, by using the fluorescence quenching technique, to selectively stain the plasma membrane of viable neutrophils.     

Sorting of micronuclei from PtK1 cells

We report here a procedure allowing to select micronuclei corresponding to defined individualized chromosomes in conditions which preserve their synthetic activity. The mammalian PtK1 cells, which possess six chromosome pairs, were micronucleated by colchicine. DNA of the micronucleated cells was labeled by the Hoechst 33342 fluorochrome under vital conditions. The micronuclei were isolated by a gentle procedure and their fluorescence was analysed by flow cytometry. The flow-cytometry parameters were determined for the analysis of non-fixed subdiploid fractions. We obtained five distinct peaks of fluorescence which have been sorted. The sorted micronuclei are different in each peak exhibiting different fluorescence intensity. Peak 3 contains the micronuclei with nucleoli and chromocenters that correspond to the X chromosome in this cell line.     

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.     

1-Phenylethynylpyrene (1-PEPy) as refined excimer forming alternative to pyrene: case of DNA major groove excimer

1-Phenylethynylpyrene fluorochrome was studied as meta- and para-derivatives of arabino-uridine-2'-carbamates in ss and dsDNA. 1-PEPy showed red-shifted emission and increased fluorescence quantum yield compared to pyrene. Although 1-PEPy has very short excited lifetime (<2.5 ns), it is able to form inter- and intrastrand excimers on DNA, probably resulting from spatial preorganization of two dye molecules.     

A simple fluorescence technique to stain the plasma membrane of human neutrophils

Summary Three different fluorochromes were tested for their ability to label the plasma membrane proteins of neutrophils without labelling intracellular structures. A fluorescence quenching technique was used to differentiate between extra- and intracellularly localized fluorescence. Fluorescamin and fluoresceinisothiocyanate were shown to stain intracellular structures as well as the plasma membranes of the cells. Another fluorochrome, Evans Blue, is proposed since this dye was shown, by using the fluorescence quenching technique, to selectively stain the plasma membrane of viable neutrophils.To whom offprint requests should be sent     

Cytofluorometric Determination of Nuclear DNA in Living and Preserved Algae

Three DNA-localizing fluorochromes used in conjunction with epi (incident) UV illumination were examined for sensitivity and selectivity for the cytofluorometric determination of nuclear DNA in ten species of six algal genera: Mougeotia, Oedogonium, Sirogonium, Spirogyra and Zygnema among the green algae, and the marine red alga Polysiphonia boldii. In comparison with absorption photometry for the determination of nuclear DNA, the cytofluorometric procedure proved to be simpler and considerably more sensitive. Following staining with 4',6-diamidino-2-phenylindole (DAPI), nuclei fluoresce blue-white, the fluorescence intensity of the DNA-DAPI complex being considerably greater than that of the unbound dye molecule. Algal strains stained with 2,5-bis[4'-aminopheny](1')]-1,3,4-oxadiazole (BAO) also showed brilliant blue-white nuclear fluorescence. Although the BAO schedule requires the use of freshly prepared dye and sulfite water, and careful control of hydrolysis, nuclear fluorescence of the stained specimens does not fade under irradiation of the UV beam as rapidly as it does with certain other fluorochrome procedures. A more useful fluorochrome was the fungal antibiotic mithramycin. Its staining schedule is simple and the bright orange-yellow fluorescence of the nuclei is associated with an exceptional degree of sensitivity and specificity for DNA. Forty-eight-year-old preserved filaments of Spirogyra jatobae, stained with either BAO or mithramycin, exhibited a fluorescence brilliance of nuclear and chloroplast DNA equal to that of fresh specimens of this species. The three schedules, but particularly the one with mithramycin, have proven useful in providing indirect evidence for variation in ploidy level in several of the above algal genera, and in verifying the assumed ploidy level of the gametophyte (haploid) and tetrasporophyte (diploid) of Polysiphonia boldii     

Detection of very low receptor numbers on cells by flow cytometry using a sensitive staining method

We describe a staining method for flow cytometry that resolves with a high degree of sensitivity very low numbers of cell surface molecules, which are normally too few to detect using the conventional fluorescein-conjugated reagents. We took advantage of the fact that liposomes can be constructed to contain hundreds of thousands of fluorochrome molecules per vesicle; antigen specificity can be conferred by covalently conjugating them to antibodies or protein A. Unlike fluorochromes such as fluorescein isothiocyanate (FITC) that are directly conjugated to protein ligands with a fluorochrome to protein ratio of about 2 to 1 on the average, their large encapsulating capacity gives liposomes a tremendous potential for signal amplification. In an indirect immunofluorescence study using liposomes that contained the fluorochrome carboxyfluorescein (CF) and that were covalently conjugated to protein A, we were able to obtain up to 50 times the fluorescence signal over background that could be detected with FITC-conjugated protein A. Scatchard analysis showed that the thymoma cell line RDM4 expresses 23,000 and 2,600 binding sites for monoclonal antibodies (mAb) against H-2K and H-2D, respectively. When RDM4 cells were treated with anti-H-2K mAb followed by FITC-conjugated protein A, at best we were able to obtain a fluorescence signal that was only 7 times above background. However, when these cells were treated with the same antibody followed by protein A conjugated to small unilamellar liposomes or large unilamellar liposomes, the fluorescence signals were 110 and 335 times above background, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)