A Fluorescent Dye with Low Specificity to DNA Nucleotide Sequences: Quantitative Assessment of Oligonucleotides Immobilized in Microchip Gel Pads

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)₈, d(T)₈, d(C)₈, and d(G)₈ at high temperatures (50°C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.     

光激活荧光蛋白及其在植物分子细胞生物学研究中的应用

光激活荧光蛋白是指用特定光照射时,其荧光特性发生显著改变的一类荧光蛋白。借助光激活荧光蛋白的这种特性,可以实现对活细胞、细胞器或胞内分子的时空标记和追踪。该文介绍了目前光激活荧光蛋白的性质,并从多个方面对其应用进行了概括,包括分子标记与动态分析、蛋白质相互作用、细胞器及细胞组分动态研究、细胞追踪以及在光激活定位显微镜中的应用等,且对目前光激活荧光蛋白在植物分子细胞生物学中的应用进行了详细介绍。     

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.     

光激活荧光蛋白及其在植物分子细胞生物学研究中的应用

光激活荧光蛋白是指用特定光照射时, 其荧光特性发生显著改变的一类荧光蛋白。借助光激活荧光蛋白的这种特性,可以实现对活细胞、细胞器或胞内分子的时空标记和追踪。该文介绍了目前光激活荧光蛋白的性质, 并从多个方面对其应用进行了概括, 包括分子标记与动态分析、蛋白质相互作用、细胞器及细胞组分动态研究、细胞追踪以及在光激活定位显微镜中的应用等, 且对目前光激活荧光蛋白在植物分子细胞生物学中的应用进行了详细介绍。     

Intracellular dye heterogeneity determined by fluorescence lifetimes

The cellular localization of a fluorescent probe molecule depends on both the chemical structure of the dye and the cellular environment. To study the number and types of environments in an epithelial cell line, we have measured in Madin-Darby canine kidney (MDCK) cells the fluorescence lifetimes of three structurally distinct fluorescent dyes — rhodamine-B, 3,3′-dihexadecylindocarbocyanine-(C₃) (diI), and Collarein — incorporated into these cells. The latter is a rhodamine-cardiolipin conjugate that we designed and synthesized for the property of exclusive localization in the plasma membrane. The former two dyes required at least two exponential components to fit their fluorescence decay curves, while the decay of Collarein was characterized by a single exponential. These data are consistent with fluorescence microscopic observations, in which diI and rhodamine-B exhibit heterogeneous spatial distributions, while Collarein appears to be located on the cell surface.     

Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.     

The use of fluorescent dyes to measure membrane potentials: a response

The use of fluorescent cyanine dyes to estimate membrane potential in cell suspensions has been considered. Several problems related tot he application of the dyes have been reviewed. These problems include: 1) alteration of the membrane potential (Em) and factors involved in establishing Em by the dyes themselves, 2) the effects of altered energy metabolism on the fluorescent response of the dyes and on Em, and 3) calibration of dye fluorescence. Recent reports that advocate the use of the fluorescent dyes are misleading.     

New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids

The spectroscopic properties of three new dyes, EK4, VL772, and LL585, free and bound to nucleic acids, are presented, with particular emphasis on their potential use in flow cytometry. Two of these dyes, EK4 and LL585, exhibit red fluorescence, while dye VL772 exhibits yellow fluorescence. Dye LL585 exhibits specificity for DNA, relative to RNA, and a marked enhancement of fluorescence efficiency upon binding to DNA, needed for a red fluorescent DNA-specific stain for flow cytometry. The dye penetrates live cells, although uniformity of nuclear fluorescence, as evidenced by DNA flow histograms, is better if the cells are first permeabilized with Triton X-100. Dye VL772 exhibits yellow fluorescence and little DNA-RNA discrimination, but may prove useful in conjunction with dye LL585 when simultaneous assay of cellular RNA and DNA is desired. Dye EK4 shares properties of the other two dyes but fluoresces with much less efficiency. Dyes LL585 and VL772, used singly, as a pair, or in combination with blue-fluorescing DNA specific dyes, such as bisbenzimidazole derivatives, should permit new, convenient analyses of the content and organization of cellular nucleic acids.     

Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent

For the evaluation of cell membrane electropermeabilization, cells are usually exposed to electric pulses in the presence of propidium iodide, a fluorescent dye activated by binding to cellular DNA. The fraction of permeabilized cells is then determined using a flow cytometer. This widely established method has several drawbacks: (i) an arbitrary choice of minimum fluorescence intensity for characterization of permeabilized cells; (ii) the inability to detect cells disintegrated because of intense electropermeabilization; and (iii) false detection of cellular ghosts devoid of fluorescence because of leakage of DNA caused by electropermeabilization. Here, we present a simple and inexpensive method that eliminates these drawbacks. The method is based on the use of a cytotoxic agent that cannot permeate through an intact plasma membrane and thus leads to selective death of the electropermeabilized cells. The amount of nonpermeabilized cells is then determined by a suitable viability test. Bleomycin at a 5-nM concentration causes no statistically significant effect on cell survival in the absence of electric pulses, yet this concentration is sufficient for lethal toxicity in electropermeabilized cells. The amount of cells surviving the exposure relative to the control gives a reliable value of the fraction of nonpermeabilized cells.     

DiOC6(3): a Useful Dye for Staining the Endoplasmic Reticulum

The present review discusses the fluorescent organelle probe, DiOC₆(3), with reference to its structure, chemistry, availability, spectral properties, labeling procedures, vital staining characteristics, and major applications in cellular and molecular biology. The specificity of dye for endoplasmic reticulum is summarized. We examine the simplicity and advantages of the fluorescent dye system for evaluating structure and function of endoplasmic reticulum. Other significant uses of the dye are also discussed.     

A versatile assay to study cellular uptake of gene transfer complexes by flow cytometry

In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i. e. binding and internalisation of transfection complexes. Taking advantage of flow cytometry, it is possible to discriminate quantitatively between total and internal DNA on a single-cell level. Here, we use two fluorescent dyes with high specificity and affinity to double-stranded DNA that cannot penetrate the extracellular membrane of living cells. Total DNA is stained prior to complexation with the first dye and complexes are added to cells. After the incubation, only extracellular DNA remains accessible to the second dye. Cell associated fluorescence is measured simultaneously using a flow cytometer and data are analysed using a computer program capable of calculating the ratio of fluorescence intensities on a single-cell level. These ratios are indicative of the binding and internalisation kinetics of gene transfer complexes.     

Influence of trans-membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells

The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for photoaffinity labelling of mitochondrial proteins. By a combination of photoaffinity labelling of cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonylcyanidephenylhydrazon (CCCP), strongly reduces or even prevents APMC labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their R _m values in reversed phase thin-layer chromatography. Prestaining with an excess of strongly lipophilic cationic acridine and phenanthridine dyes, such as pentyl acridinium orange chloride (PAO), nonyl acridinium orange chloride (NAO) and tetramethylpropidium chloride (MP), respectively, completely prevents protein labelling with APMC. Obviously, the dyes occupy the same mitochondrial binding sites as APMC. At equal concentrations the intensity of the 40-kDa signal is strongly reduced, whereas the 31-kDa and 74-kDa signals are unaffected. Using phenanthridine dyes with lower lipophilicity, namely propidium chloride (P), M, and N reduces the peak of the 40-kDa protein in APMC labelling, indicating that the 40-kDa protein preferentially binds lipophilic dye cations.     

Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".     

Microstructured liposome array

Conversion of a DNA chip to a nanocapsule array was performed by grafting on a liposome an oligonucleotide complementary to an oligonucleotide bound to the array. Each liposome may be loaded by a soluble molecule or may present a hydrophobic or amphiphilic molecule inserted in its wall. To detect liposomes on the chip, we used fluorescent dyes encapsulated in the liposome internal volume or fluorescent lipids. We observed that an oligonucleotide-grafted liposome containing a defined dye specifically accumulated on the area where its complementary oligonucleotide had been spotted on the array. The virtually unlimited amount of addresses allows the specific binding of large amounts of liposomes in one single batch.     

Exchange of counterions in DNA condensation

We measured the fluorescence intensity of DNA-bound fluorescent dyes YO-PRO-1 (oxazole yellow) and YOYO-1 (dimer of oxazole yellow) at various spermidine concentrations to determine how counterions on DNA are exchanged in the process of DNA condensation. A decrease of fluorescence intensity was observed with an increase of spermidine. Considering the chemical equilibrium under the competition between the dye and spermidine for counterion condensation on DNA, the theoretical curve well describes the decrease of the fluorescence intensity. These results indicate that dyes are exchanged for spermidine at the binding site on DNA; that is, the exchange of counterions occurs. The parameters associated with the decrease of the fluorescence intensity show that the relative affinity of the dye and spermidine for DNA depends on the state of DNA. Moreover, YOYO-1 prevents the DNA condensation, but the effect of YO-PRO-1 on the condensation is very slight, though both dyes intercalate for DNA; the high affinity of YOYO-1 compared to YO-PRO-1 enables prevention of the condensation.     

Fluorescent target array killing assay: a multiplex cytotoxic T-cell assay to measure detailed T-cell antigen specificity and avidity in vivo

Here we describe a multiplex, fluorescence-based, in vivo cytotoxic T-cell assay using the three vital dyes carboxyfluorescein diacetate succinimidyl ester, cell trace violet, and cell proliferation dye efluor 670. When used to label cells in combination, these dyes can discriminate >200 different target cell populations in the one animal due to each target population having a unique fluorescence signature based on fluorescence intensity and the different emission wavelengths of the dyes. This allows the simultaneous measurement of the in vivo killing of target cells pulsed with numerous peptides at different concentrations and the inclusion of many replicates. This fluorescent target array killing assay can be used to measure the fine antigen specificity and avidity of polyclonal cytotoxic T-cell responses in vivo, immunological parameters that were previously impossible to monitor.     

An Improved Method for Studying Microvascular Geometry Using Fluorescent Dyes: Preventing Dye Extravasation, Preserving Dye Integrity and Enhancing Tissue Morphometry

A procedure for stabilizing fluorescent markers used to study the microvascular geometry and morphometry of muscle tissue is described. The procedure involves fluorescent labeling of plasma. fixation of muscle tissue in 10% buffered formalin. and quick freezing. This procedure prevents extravasation of the fluorescent dyes out of the capillaries as frequently seen in other muscle microvascular techniques, thereby greatly increasing the time that capillaries are visible. We found that formalin may actually increase the rate of fluorochrome bleaching by photo-oxidation, but the increased rate of bleaching is more than offset by the greater concentration of dye trapped in the capillaries. Further, formalin fixation results in little distortion of the muscle fibers themselves, making this approach ideal for morphometric studies.     

Fluorescent dyes for lymphocyte migration and proliferation studies

Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long-term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, both in vitro and in vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short-term (up to 3 day) lymphocyte migration experiments, namely the DNA-binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.     

Characterization of six textile dyes as fluorescent stains for flow cytometry.

Few fluorescent stains specific for cell constituents other than DNA are available. To assess their potential use as fluorescent stains for flow cytometry, the cell staining specificity of 55 compounds, originally synthesized for use as textile dyes and fluorescent brighteners, was explored and their excitation and emission wavebands determined. From these, six dyes were chosen for more detailed analysis. All six are vital stains, with excitation wavelengths allowing their use with an argon ion laser, and specific for a range of cell structures including mitochondria, Golgi bodies, lipid droplets, nuclear membrane, and endoplasmic reticulum. Concentrations as low as 0.01-0.25 microM were found to be adequate for most purposes, and high background fluorescence was not a problem. Their specificity allows differentiation between non-cycling and cycling cells. The properties of two of the stains allows their combination with propidium iodide or ethidium bromide for simultaneous determination of DNA content profiles. Being vital stains, usable at very low concentrations, and specific for a range of cell organelles, these six stains may be of considerable utility in flow cytofluorometry. We suggest that other textile dyes may be of use in flow cytofluorometry, or that their structures may form a starting point for the synthesis of further fluorescent stains of enhanced specificity.     

Dye with low specificity to nucleotide sequences of DNA: use for assessing the quantity of oligonucleotides, immobilized in cells of biological microchips

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50 degrees C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.