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PubMed1994

A simple fluorometric assay for the determination of cell numbers.

A sensitive fluorometric assay was developed for the enumeration of cells in microtiter plates. This assay is based on the fluorescence enhancement of propidium iodide (PI) upon binding with double-stranded nucleic acids. This fluorochrome is compatible with a wide range of reagents commonly used in the laboratory, thus washing the cells before staining is not necessary. PI, together with Triton-X 100 and EDTA, was added directly to the cell culture. After 16-18 h incubation at room temperature, intensity of fluorescence was determined with a micro-plate fluorometer. This quick and simple method is sensitive for as little as 1.95 x 10(3) mononuclear leukocytes, and provides a linear correlation (r = 0.999) between cell number and fluorescence up to 1 x 10(6) cells. Since PI has a large Stokes shift with excitation wavelength at common visible range and emission wavelength far out in the red region of the spectrum, it allows simultaneous detection of DNA and other fluorescent compounds such as calcein and fluorescein. This assay may prove to be a valuable alternative for cell number determination.

Animals↗
PubMed1989

Clinical evaluation for the mechanism of cisplatin with bromodeoxyuridine by flow cytometry and clinico-histological study.

The tumor cell kinetics before and after intra-arterial injection of cisplatin (CDDP) was examined in a patient with maxillary carcinoma using flow cytometry and propidium iodide and fluorescein isothiocyanate labeled bromodeoxyuridine double staining. In addition, the viability was determined with fluorescein diacetate and compared with the histological picture. The action mechanism which the authors obtained 24 hours after intra-arterial injection of CDDP to the clinical case was considered to be S phase block, especially early S phase block. Markedly decreased viability also makes us consider the cytocidal effect. These findings correlated with the data of the fundamental investigation reported previously. Histological examination after the intra-arterial injection of CDDP revealed remarkable necrosis in the tissue.

Bromodeoxyuridine↗
PubMed1993

A quantitative evaluation of erythropoiesis in myelodysplastic syndromes using multiparameter flow cytometry.

By staining human bone marrow cells with a monoclonal antibody reacting with erythroid precursor cells (AS-E1) and propidium iodide, we have evaluated the proliferative capacity of erythropoiesis in patients with myelodysplastic syndromes (MDS) using flow cytometry. Comparing 36 patients (13 RA/RAS, 13 RAEB, 10 RAEB-t) with 7 normal controls, significant differences in both the percentage of AS-E1+ cells and the fraction of AS-E1+ cells in the S or S-G2M-phase between the four groups were found. Since neither the percentage of AS-E1+ cells nor their fraction in S or S-G2M alone was found to characterize their proliferative activity, we introduced the proliferative fractions of the erythroid cell, i.e. the number of the AS-E1+ cells in S or S-G2M related to all bone marrow cells in S or S-G2M. Applying these parameters, we found significantly increased proliferative AS-E1 fractions in the RA/RAS group compared to the normal controls (p = 0.03 and 0.002) respectively, as well as a highly significant decrease with disease progression.

Antibodies, Monoclonal↗
PubMed1986

The use of DNA-intercalating dye to monitor cell fusion and microinjection.

A conventional method for microinjection, using erythrocyte ghosts as the injection vector, has been modified to provide a protocol for the highly efficient delivery of small quantities of material into the cytoplasm of target cells. The technique is applicable for use with a variety of proteins, sugars, nucleotides and dyes. When the intercalating dye propidium iodide is included within the sealed ghosts their subsequent fusion with target cells can be continuously monitored by fluorescence spectroscopy, providing a convenient and sensitive parameter of cell-cell fusion. The protocol can be adapted for use with both adherent and non-adherent target cells, and can be used to monitor the relative effectiveness of a variety of fusogenic agents.

Cell Fusion↗
PubMedDate not supplied

Quadruple labeling of brain-stem neurons: a multiple retrograde fluorescent tracer study of axonal collateralization.

Four different fluorochromes were injected into adjacent cervical spinal cord segments, 1 unique tracer per segment. Each tracer, Fluoro-Gold, Fast Blue, Diamidino Yellow dihydrochloride and Propidium Iodide, was taken up by axonal terminals and transported intra-axonally in a retrograde direction to the cell bodies. Some, though by no means all, of these axons were stem axons with terminals in 2, 3 or 4 of the injected spinal segments. Hence as many as 4 different fluorescent tracers could be discerned simultaneously within individual neuronal somata of origin using fluorescent microscopy. These results extend the possibilities for multiple interconnection determinations within the central nervous system. Specifically, the potential for individual neurons of a nucleus to project collateral branches of a stem axon to as many as 4 different central nervous system nuclei now can be studied simultaneously using these 4 fluorescent tracers.

Afferent Pathways↗
PubMedDate not supplied

Electron microscopic analysis of fluorescent neuronal labeling after photoconversion.

Ultrastructural visualization of non-electron-dense fluorescent retrograde neuronal labeling was attempted by means of photo-oxidation. This procedure was used to convert the fluorescence of neurons labeled by the tracers propidium iodide, rhodamine latex microspheres and fluorogold into a stable diaminobenzidine reaction product. The ultrastructural study revealed an accumulation of electron-dense material in these cells both within lysosomes and scattered in the cytoplasmic matrix. Comparison with several different sets of control samples indicated that this material, on the basis of its amount, electron density and appearance, specifically represents the photoconversion reaction product. The effects of the intensity of the fluorescent labeling and of a prolonged photoconversion on the fine structural features of the reaction product are also described and discussed. The present findings indicate that photoconversion can be effectively applied to ultrastructural study of fluorescent retrogradely labeled neurons. The specificity of the photoconversion reaction product should be tested routinely for each fluorochrome and tissue sample.

Amidines↗
PubMed1994

Measurement and characterization of micronuclei in exfoliated human cells by fluorescence in situ hybridization with a centromeric probe.

The micronucleus (MN) assay in human exfoliated cells has been widely used to detect the genotoxic effects of environmental mutagens, infectious agents and hereditary diseases. Substantial variability characterizes the MN frequencies reported by different research groups. One reason for this may be the restricted resolution power of the Feulgen-Fast-Green staining that is routinely used. Here we describe a new version of the MN assay that employs fluorescent propidium iodide staining along with fluorescence in situ hybridization (FISH) with a centromeric probe. Buccal and urothelial cells were collected from 5 healthy unexposed female volunteers and 55,000 cells analyzed for MN frequency and abnormal nuclear events. The Feulgen-Fast-Green and the new fluorescent staining produced very similar results. The frequency of MN in buccal cells was 0.145 +/- 0.118% and in urothelial cells 0.083 +/- 0.074%. No correlation was found between the frequencies of MN in the two types of exfoliated cells. FISH with a centromeric probe allowed MN containing whole chromosomes with a centromere to be differentiated from those containing only acentric fragments. The former appear as a result of chromosome lagging in mitosis, while those without a centromere are due to chromosome breakage. In urothelial cells 43% of MN were centromere-negative and in buccal cells -44%. Fluorescent staining provided more accurate scoring of degenerative cells than standard Feulgen-Fast-Green staining. The combined frequency of pycnotic cells, "broken eggs" and cells with fragmented nuclei did not exceed 2%, while that of karyorrhexis and karyolysis together was as high as 21%. Significant interindividual variability was found in the frequency of cells with karyolysis and karyorrhexis. Thus, the new version of micronucleus assay allows for MN to be scored more precisely, the mechanism of MN formation to be determined and abnormal nuclear events to be readily identified in exfoliated human cells. It is therefore ideal for studying genotoxicity in human populations using exfoliated cells from the mouth, bladder and nose.

Adult↗
PubMed1995

A new fluorochromasia method using a fluorescence microplate reader for assay of cytotoxic activity of carp leucocytes.

A new fluorochromasia method using a fluorescence microplate reader has been established to assay spontaneous cytotoxic activity of carp leucocytes. This method is characterized by using propidium iodide (PI) for staining dead target cells and a fluorescence microplate reader for measurement of the fluorescence of PI. K562 as target cells were prepared in 96-well flat-bottom microplates, and carp leucocytes were added as effector cells. After 2.5 h incubation, PI was added to each well. After an additional 1.5 h incubation, fluorescence of each well was measured. Correlation between this method and 51Cr-release assay was obtained. The results demonstrated that this new fluorochromasia method can be used to assay cytotoxic activity of carp leucocytes.

Animals↗
PubMed1993

Altered lipid packing identifies apoptotic thymocytes.

To test whether apoptotic thymocytes can be identified by altered packing of lipids in their plasma membranes, thymocytes were isolated from mice injected with hydrocortisone and stained with merocyanine 540 (MC540), a fluorescent probe sensitive to lipid packing. At 9-10 h after injection, a subpopulation of H2-Klo cells with increased MC540 staining was clearly discernable. When DNA degradation was assessed by staining fixed cells with propidium iodide (PI), the fraction of cells with increased MC540 staining corresponded to the fraction with reduced PI staining. In addition, enriching for the former by fluorescence-activated cell sorting enriched for the latter. These results indicate that living apoptotic thymocytes can be identified and separated on the basis of altered lipid packing and increased staining with MC540.

Animals↗
PubMed1991

p-Butyroxybenzenediazonium fluoroborate, substrate of acetylcholinesterase and butyrylcholinesterase, discriminates between the two enzymes by a specific affinity labelling.

p-Butyroxybenzenediazonium fluoroborate 1 was shown to be a substrate of both acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE) with Michaelis constants of 6.10(-5) M and 1.3. 10(-4)M, respectively. Upon incubation in the dark, 1 was able to discriminate between the two enzymes AcChE was efficiently inactivated in a time-dependent manner while BuChE remained unaffected. Kinetic analysis of the inactivation of AcChE (i) by various concentrations of 1 indicated that it behaves as an affinity label, (ii) at three different pH levels suggested that the pKa of the labelled residue was higher than 7 and (iii) in the presence of different selective ligands for either the active site (edrophonium) or the peripheral site (propidium) indicated that 1 alkylated the active site rather than the peripheral one. Differences of reactivity between AcChE and BuChE suggest a different positioning and/or a different chemical environment of the substrate within two active sites.

Acetylcholinesterase↗
PubMed1992

Evaluation of an automated method of percent reactive antibody determination.

A fluorescence-based automated method of percent reactive antibody (PRA) analysis is described. This method utilizes the conventional antibody-mediated, C'-dependent lymphocyte microcytotoxicity assay to detect alloantibodies, but replaces the eosin-based method for detection of cell death with a fluorescence-based method. To identify viable cells, lymphocytes were pretreated with carboxy fluorescein diacetate (CFDA), which fluoresces green, to identify viable cells. To identify dead cells after the reaction with antibody and C', they were treated with propidium iodide (PI), which fluoresces red. Pretreatment of lymphocytes with CFDA did not affect their ability or interact with alloantibodies in the microcytotoxicity assays. When visually analyzed, detection of cell death by fluorescence was as sensitive as detection by eosin exclusion. However electronic detection of fluorescence was slightly more sensitive than visual detection. Automation of the fluorescent method required a calculation that converts electronic data to an ASHI score for cell death. One such method is described and evaluated. Both the automated and the conventional methods of analysis were used to obtain PRA values for various sera. There was good correlation between the PRA values obtained with the automated method versus the conventional method. Further, there was good correlation for PRA-derived alloantibody specificities obtained with the automated method versus the conventional method. These data demonstrate that automated fluorescence-based PRA analysis is an effective and practical alternative to conventional PRA analysis.

Antibodies↗
PubMed1993

A digitized fluorescence imaging study of intracellular free calcium, mitochondrial integrity and cytotoxicity in rat renal cells exposed to ionomycin, a calcium ionophore.

The objective of this study was to explore the role of extracellular Ca2+ and mitochondrial integrity in ionomycin-induced cytotoxicity in primary cultures of rat kidney cortical epithelial cells using digitized fluorescence imaging (DFI), which is a powerful tool for continuously observing the dynamic intracellular biochemistry of single living cells. Using DFI, intracellular free calcium ion concentration ([Ca2+]i), mitochondrial membrane potential and loss of cell viability in individual rat renal cortical epithelial cells were examined temporally by fura-2, rhodamine 123 (Rh-123) and propidium iodide (PI), respectively. Images were taken within 10 min after exposure to 5 and 10 microM ionomycin. These three parameters, [Ca2+]i, mitochondrial membrane potential and cell viability, were also measured in populations of cells by a multiwell fluorescence scanner with fluo-3, Rh-123 and PI, respectively. Cytotoxicity was also assessed by two colorimetric cytotoxicity tests (LDH leakage and mitochondrial MTT reduction). Using DFI, the fluorescence scanner and the colorimetric cytotoxicity tests, we found that exposure of primary cultures of rat kidney cortical epithelial cells to high concentrations of ionomycin (5 and 10 microM) caused a rapid and sustained rise in [Ca2+]i, which preceded dissipation of the mitochondrial membrane potential and loss of cell viability and that chelation of extracellular Ca2+ with EGTA attenuated these responses. We demonstrated the value of using DFI to continuously observe the dynamic intracellular biochemistry of single living cells by establishing a sequence of elevated [Ca2+]i, dissipation of mitochondrial membrane potential and cytotoxicity. We conclude that a combination of the influx of extracellular Ca2+ and loss of mitochondrial integrity may be responsible for the cytotoxicity observed in individual renal cells and populations of renal cells after treatment with ionomycin.

Aniline Compounds↗
PubMed1987

Nonspecific interactions in dye binding to DNA. Influence of alcohols and amides.

The binding of a few drugs (ethidium bromide, propidium diiodide, proflavine and actinomycin D) to DNA has been investigated in aqueous solutions to which cosolvents of different polarity have been added. It is found that both alcohols (less polar than water) and amides (more polar) lower the binding constant according to a linear relationship between the intercalation free energy and cosolvent concentration. The main action of cosolvents cannot be described in terms of electrostatic effects, since they predict much smaller changes in the binding constant than those observed. It appears instead that relevant solvation effects are responsible for the binding strength of the different dyes to DNA. As a general result, it is found that solvation effects largely contribute to the intercalation free energy, thereby weakening the influence of nonspecific interactions at the intercalation site.

Alcohols↗
PubMed1995

Flow cytometric analysis of cell suspensions exposed to shock waves in the presence of the radical sensitive dye hydroethidine.

The occurrence of intracellularly and extracellularly generated free radicals during shock wave exposure on an experimental Siemens lithotripter was tested with the radical sensitive dyes hydroethidine and dichlorofluorescin (DCFH). DCFH, a nonfluorescent compound, is oxidised to dichlorfluorescein (DCF) by hydrogen peroxide in the presence of peroxidase. DCF green fluorescence intensity was used for fluorescence spectrometric measurement of hydrogen peroxide generated during shock-wave treatment of cell-free dye solutions. The fluorescence intensity of ethidium, the oxidised form of hydroethidine, was used for the flow-cytometric measurement of intracellular oxidising reagents present in RT4 tumour cells during shock-wave exposure. Changes in membrane permeability, which influence the intracellular content of ethidium, were controlled by counterstaining the cells with propidium iodide, an indicator for membrane integrity. We observed no increase in intracellular ethidium fluorescence intensity after shock-wave treatment of single cell suspensions and therefore no indication for shock-wave-induced intracellular free radicals.

Carcinoma, Papillary↗
PubMed1980

Microiontophoretic injection of fluorescent tracer combined with simultaneous immunofluorescent histochemistry for the demonstration of efferents from the caudate-putamen projecting to the globus pallidus.

Precise local injections of propidium iodide (PI) were made, using an iontophoretic technique, into various regions of the globus pallidus (GP) of rats. The locations of the injection sites and the position of retrogradely labelled caudate putamen (CP) cell bodies demonstrated a topographic arrangement of efferents from the CP projecting to the pallidum. The PI labelled cell bodies in the CP were also counterstained for met-enkephalin immunofluorescence. Cell bodies labelled with PI, which showed co-existing enkephalin immunofluorescence, were found in CP projections to the pallidum.

Animals↗
PubMed1981

Single preganglionic sympathetic neurons of the cat branch intraspinally and project through different rami communicantes albi--a retrograde double labelling study with fluorescent tracers.

Evidence is presented that in lumbar segments of the cat, two neighbouring rami communicantes albi (white rami, WR) may contain axons of one and the same preganglionic sympathetic neurone (PSN). This was shown with bisbenzimide (Bb) and propidium iodide (PI) applied in the same cat to the central cut ends of two WR (PI to WRL1, Bb to WRL2). PSN, double labelled from both these sites, were observed in the intermediolateral and intercalate nuclei of the L1 segment. It is postulated that axons of PSN branch intraspinally and project through different WR.

Animals↗
PubMed1990

Photoconversion of fluorescent retrograde tracers.

Photoconversion of fluorescent dyes, retrogradely transported through axons to their parent cell bodies, into a stable diaminobenzidine (DAB) reaction product was tested in the nigrostriatal and thalamocortical systems of rats. Satisfactory results were obtained with Propidium Iodide (PI), Fluoro-Gold (FG), Fast Blue (FB), Diamidino Yellow (DY), and rhodamine-labeled latex microspheres (RLM); some photoconversion was also observed in Evans Blue (EB)-labeled neurons. The red fluorescent tracers PI, EB and RLM were photoconverted under the excitation wavelength appropriate for eliciting their fluorescent emission. With the yellow or blue fluorescent tracers FG, FB, and DY satisfactory results could instead be obtained using an excitation wavelength which did not elicit visible fluorescent emission. This finding indicates that the latter is not a critical factor for obtaining photoconversion. The features and subcellular localization of photoconverted DAB were different from those of the fluorescent labeling: photoconversion resulted in the appearance of brown granules of DAB reaction products in the cytoplasm, independently from the occurrence of fluorescent labeling in the neuronal cytoplasm or nucleus. Photoconversion may enable new applications of fluorescent retrograde tracing and, in particular, its electron microscopic visualization.

Animals↗
PubMed1990

Collateral projections from the midbrain periaqueductal gray to the nucleus raphe magnus and nucleus accumbens in the rat. A fluorescent retrograde double-labelling study.

Collateral projections of single neurons in the midbrain periaqueductal gray (PAG) to the nucleus accumbens (ACB) and nucleus raphe magnus (NRM) were observed in the rat by a fluorescent retrograde double-labelling technique. After injecting propidium iodide into the ACB and bisbenzimide into the NRM, doubly labelled PAG neurons were most frequently seen in the ventrolateral subnucleus and ventral part of the medial subnucleus at the middle and caudal levels of the PAG.

Animals↗