Highly fluorochrome labeled gene probes for quantitative tracing of RNA in individual cells by in situ hybridization.

A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochrome-labeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeled gene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation.     

Quantitative characterization of single‐cell adhesion properties by atomic force microscopy using protein‐functionalized microbeads

A method was developed to characterize the adhesion properties of single cells by using protein‐functionalized atomic force microscopy (AFM) probes. The quantification by force spectroscopy of the mean detachment force between cells and a gelatin‐functionalized colloidal tip reveals differences in cell adhesion properties that are not within reach of a traditional bulk technique, the washing assay. In this latter method, experiments yield semiquantitative and average adhesion properties of a large population of cells. They are also limited to stringent conditions and cannot highlight disparities in adhesion in the subset of adherent cells. In contrast, this AFM‐based method allows for a reproducible and quantitative investigation of the adhesive properties of individual cells in common cell culture conditions and allows for the detection of adhesive subpopulations of cells. These characteristics meet the critical requirements of many fields, such as the study of cancer cell migratory abilities.     

A novel LED‐based 2D‐fluorescence spectroscopy system for in‐line monitoring of Chinese hamster ovary cell cultivations – Part I

A new two‐dimensional fluorescence sensor system was developed for in‐line monitoring of mammalian cell cultures. Fluorescence spectroscopy allows for the detection and quantification of naturally occurring intra‐ and extracellular fluorophores in the cell broth. The fluorescence signals correlate to the cells’ current redox state and other relevant process parameters. Cell culture pretests with twelve different excitation wavelengths showed that only three wavelengths account for a vast majority of spectral variation. Accordingly, the newly developed device utilizes three high‐power LEDs as excitation sources in combination with a back‐thinned CCD‐spectrometer for fluorescence detection. This setup was first tested in a lab design of experiments study with process relevant fluorophores proving its suitability for cell culture monitoring with LOD in the μg/L range. The sensor was then integrated into a CHO‐K1 cell culture process. The acquired fluorescence spectra of several batches were evaluated using multivariate methods. The resulting batch evolution models were challenged in deviating and “golden batch” validation runs. These first tests showed that the new sensor can trace the cells’ metabolic state in a fast and reliable manner. Cellular distress is quickly detected as a deviation from the “golden batch”.     

Evaluation of different methods detecting intracellular generation of free radicals

Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn²⁺. All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.     

A multiple-staining procedure for the detection of different DNA fragments on a single blot.

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometry analysis of DNA degradation in thymocytes of gamma-irradiated or hydrocortisone treated rats

The pattern of DNA degradation in thymocytes of irradiated or hydrocortisone-treated rats has been studied by means of flow cytometry of the cells, treated with probes specifically bound to the AT or GC-pairs of DNA. It has been shown that the death of thymocytes is accompanied by a decrease in their DNA content. The main features of the occurrence and accumulation of cells with a DNA content less than the normal diploid level correspond with those of internucleosomal DNA fragmentation: such cells appear after a 1 hour lag-period, their accumulation is prevented by cycloheximide injection and is lower at 300 Gy than at doses of 10 to 30 Gy. At the same time, no increase in permeability of the cell membrane to ethidium bromide was observed up to the sixth hour after irradiation. Most of the thymocytes dying under the action of irradiation or hydrocortisone are in the G0 or G1 phases of the cell cycle. The method used allows detection of the cells with cleaved but not removed DNA.     

Direct‐geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms

Although fluorescence in situ hybridization (FISH) with specific ribosomal RNA (rRNA)‐targeted oligonucleotides is a standard method to detect and identify microorganisms, the specific detection of genes in bacteria and archaea, for example by using geneFISH, requires complicated and lengthy (> 30 h) procedures. Here we report a much improved protocol, direct‐geneFISH, which allows specific gene and rRNA detection within less than 6 h. For direct‐geneFISH, catalyzed amplification reporter deposition (CARD) steps are removed and fluorochrome‐labelled polynucleotide gene probes and rRNA‐targeted oligonucleotide probes are hybridized simultaneously. The protocol allows quantification of gene copy numbers per cell and the signal of the directly labelled probes enables a subcellular localization of the rRNA and target gene. The detection efficiencies of direct‐geneFISH were first evaluated on Escherichia coli carrying the target gene on a copy‐control vector. We could show that gene copy numbers correlated to the geneFISH signal within the cells. The new protocol was then applied for the detection of the sulfate thiolhydrolase (soxB) genes in cells of the gammaproteobacterial clade SUP05 in Lake Rogoznica, Croatia. Cell and gene detection efficiencies by direct‐geneFISH were statistically identical to those obtained with the original geneFISH, demonstrating the suitability of the simpler and faster protocol for environmental samples.     

A minibead method for detection of membrane lectins

Membrane lectins are being increasingly implicated in many biological phenomena. Previous methods for detection of these substances are applicable only to homogeneous cell populations. We have now developed a method that permits morphological identification of lectin-bearing cells in heterogeneous cell populations. Amide-modified latex minibeads (0.345 or 0.532 micron) were activated with glutaraldehyde and then covalently bound to p-aminophenyl derivatives of various sugars. When the probe thus constructed was incubated with cell systems known to bear well-defined membrane lectins (galactosyl receptors in hepatocytes, mannosyl receptors in macrophages), binding occurred and could be visualized by scanning electron microscopy. Binding was inhibited in the presence of excess soluble sugar, indicating the specificity of reaction. Incubation of a mixture of two different-sized probes with two different cell types led to segregation of the probes. This method also permits semiquantification of binding.     

An immunoblot assay for the simultaneous quantification of several antigens

A radioimmunologic assay method that allows for the simultaneous quantification of several antigens in one sample is described. Polypeptide antigens are resolved electrophoretically and electroblotted to nitrocellulose. The nitrocellulose is then reacted with a mixture of several antisera simultaneously, and antibody-binding proteins are visualized by incubation with 125I-protein A and by autoradiography. Antigens are identified according to their molecular weights and quantified by counting the bound radioactivity. The sensitivity of the assay is in the low nanogram range and can be adjusted individually for each antigen by appropriately diluting the first antiserum. The procedure is presently applied to the detection of three neural antigens, neural cell adhesion molecule, neuron-specific enolase, and synaptophysin, in adult brain tissue and to the assessment of expression of the latter two during development of brain cells in primary culture. The method is fast, comparatively cheap, and associated with a low radiation exposure. It should prove especially useful when only scarce amounts of sample are available.     

Inter‐laboratory validation of a rapid assay for the detection and quantification of Legionella spp. in water samples

Aims: To compare the standard culture method with a new, rapid test (ScanVIT‐Legionella?) using fluorescently labelled gene probes for the detection and enumeration of Legionella spp. The new technique was validated through experiments conducted on both artificially and naturally contaminated water and through an inter‐laboratory comparison. Methods and Results: All samples were processed by the ScanVIT test according to the manufacturer’s instructions and by a culture method (ISO 11731). ScanVIT detected significantly more positive samples, although concentrations were similar and a strong positive correlation between the two methods was observed (r = 0·888, P < 0·001). The new test was more accurate in identifying the co‐presence of Legionella pneumophila and Leg. non‐pneumophila. ScanVIT showed a slightly higher Legionella recovery from water samples artificially contaminated with Leg. pneumophila alone or together with Pseudomonas aeruginosa. Lastly, the inter‐laboratory comparison revealed that the ScanVIT test exhibits a lower variability than the traditional culture test (mean coefficient of variation 8·7 vs 16·1%). Conclusions: The results confirmed that the ScanVIT largely overlaps the reference method and offers advantages in terms of sensitivity, quantitative reliability and reduced assay time. Significance and Impact of the Study: The proposed method may represent a useful validated alternative to traditional culture for the rapid detection and quantification of Legionella spp. in water.     

Newin vitro fluorimetric microtitration assays for toxicological screening of drugs

Flow cytometry has been widely used to quantify fluorescent probes in cell culture. However, FCM is not adapted to toxicological screenings due to the cost, the length and the poor reproducibility of this technique. Moreover, several multicenter studies have preferred microtitration methodologies for drug screening. A new fluorimetric technology has been designed that is sensitive and adapted to direct screening in 96-well microplates. This fluorimeter uses cold light technology (CLF) with chemical and physical modifications of the lighting system (Rat et al., 1995). CLF allows reading of UV, visible and near infrared fluorescence by increasing light energy (from 1000 to 2300 lumens) and reducing the calorific part of light (IR>900 nm, Joule effect). It induces a decrease in background and a 500- to 1000-fold improvement of detection limit of probes in comparison with classical fluorimeters and permits detection of pg/ml to fg/ml. CLF allows easy evaluation of cell injury induced by physical agents (UVA) or chemical toxins (CCl₄). Four biological endpoints for cytotoxicity evaluation have been tested with several probes: proliferation (H33258); viability (fluorescent Neutral Red); cell-cell adhesion (calcein-AM); and mitochondrial metabolic effects (Rhodamine 123). Rh 123 assay appeared more sensitive than fluorimetric or photometric detection of Neutral Red assay. Cold light fluorimetry (CLF) permits direct detection of low concentrations of probes (pg/ml to fg/ml). CLF is shown to improve classical cytotoxicity assays and, owing to its adaptability to microtitration (in 6-, 12- or 96-well plates and in Petri dishes), it is thus a promising alternative to flow cytometry for drug cytotoxicity screening.Abbreviations CLF cold light fluorimetry - FCM flow cytometry - H33258 Hoechst 33258 - IR infrared - NIR near infrared - UV ultraviolet - MTT tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide) - Rh123 Rhodamine 123     

Detection of proteins using a colorimetric bio-barcode assay

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).     

Fluorescent oligonucleotide rDNA probes for specific detection of methane oxidising bacteria

Oligonucleotide probes targeting the 16S rRNA of distinct phylogenetic groups of methanotrophs were designed for the in situ detection of these organisms. A probe, MG-64, detected specifically type I methanotrophs, while probes MA-221 and MA-621, detected type II methanotrophs in whole cell hybridisations. A probe Mc1029 was also designed which targeted only organisms from the Methylococcus genus after whole cell hybridisations. All probes were labelled with the fluorochrome Cy3 and optimum conditions for hybridisation were determined. Non-specific target sites of the type I (MG-64) and type II (MA-621) probes to non-methanotrophic organisms are highlighted. The probes are however used in studying enrichment cultures and environments where selective pressure favours the growth of methanotrophs over other organisms. The application of these probes was demonstrated in the detection of type I methanotrophs with the MG-64 probe in an enrichment culture from an estuarine sample demonstrating methane oxidation. The detection of type I methanotrophs was confirmed by a 16S rDNA molecular analysis of the estuarine enrichment culture which demonstrated that the most abundant bacterial clone type in the 16S rDNA library was most closely related to Methylobacter sp. strain BB5.1, a type I methanotroph also isolated from an estuarine environment.     

Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips

Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample. These analyses confirmed the specificity of the assay.     

Visualization of antigenic proteins on Western blots

A new technique for the detection of antibodies bound to proteins blotted onto nitrocellulose paper was developed. The method is rapid, sensitive, and does not require radioactive probes. Proteins transferred to nitrocellulose paper are first reacted with primary antibody followed by reaction with an alkaline phosphatase conjugated second antibody. The phosphatase activity is then visualized using an agar gel impregnated with the histochemical phosphatase stain 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (J. P. Horwitz, J. Chua, M. Noel, J. T. Donatti, and J. Freisler (1966) J. Med. Chem. 9, 447; Sigma Chemical Co., Technical bulletin No. 710-EP (1978]. Antigen-antibody complexes give rise to sharp, permanent blue stained bands both on the nitrocellulose paper and in the agar overlay gel. This procedure allows detection of bands containing less than 20 ng of protein.     

Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.     

CR2 units of CR2 complexes are possibly associated with nucleophilic agents through reactive covalent links

The human complement receptor type 2 (CR2/CD21), a transmembrane glycoprotein, associates with a variety of surface antigens and proteins in the cell membrane. We examined the possibilities that the CR2 units of CR2 complexes are associated through internal covalent links reactive with nucleophilic agents, e.g. H(2)O or methylamine, and that CR2-positive cells process anti-CR2 monoclonal antibodies (MoAbs). Data from immunoblotting and cytofluorimetry with CR2-binding site-specific MoAbs show that: (i) CR2-positive Raji cells release soluble CR2 isoforms into the medium when incubated in phosphate buffered saline; (ii) despite affecting the detection of one soluble CR2 isoform, methylamine treatment of soluble CR2 allows the detection of another of its isoforms; (iii) limited pre-treatment of cells with methylamine reveals a more heterogeneous CR2-positive cell population or enhances the detection of CR2; (iv) cell treatment with CR2-binding site-specific MoAbs enhances the detection of CR2 isoform(s). The data suggest that CR2 is shed mainly as a soluble CR2 complex, in which the CR2 units link covalently and react with nucleophilic agents. Raji cells may process bound fragments (145 kDa) that are recognised by and become bound by anti-CR2 MoAb.