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PubMed1988

Rapid assessment of islet viability with acridine orange and propidium iodide.

A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential.

Acridine Orange↗
PubMed1990

Study of propidium iodide binding to DNA in intact cells by flow cytometry.

We studied the in situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by "best-fitting" of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of the in situ chromatin turned out to be reduced with respect to the non in situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow-cytometric technique for probing DNA structure in intact cells.

Binding Sites↗
PubMed2005

Propidium iodide and PicoGreen as dyes for the DNA fluorescence correlation spectroscopy measurements.

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe-nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.

DNA↗
PubMed1985

Propidium iodide and S1 nuclease: tools for studying DNA reassociation kinetics.

A method for the determination of the amount of double-stranded DNA in a reassociation mixture is described. Reassociated DNA resistant to S1 nuclease digestion is measured fluorometrically using propidium iodide. A direct comparison is made between this method and an established method in which radiolabeled Escherichia coli DNA resistant to S1 digestion is measured by scintillation counting after separation of nucleotides by Sephadex G-100 chromatography. Reassociation curves determined for calf thymus and E. coli DNA are presented.

Animals↗
PubMed1992

Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.

Animals↗
PubMed1989

Selective labeling by propidium iodide injected into the lateral cerebral ventricle of the rat.

Injections of propidium iodide (PI) into the lateral cerebral ventricle of the rat resulted in a bilateral labeling in the septohippocampal nuclei, substantia nigra (SN), ventral tegmental area (VTA), retrorubral nuclei (rr), dorsal and median raphe nuclei, regions within and dorsal to the medial lemniscus of the caudal midbrain, and Purkinje cells in the cerebellum. No labeled neurons were seen in other areas of the brain. The data suggest that PI appears to exhibit selective labeling, and the mechanism underlying the selective labeling is discussed. Combined with Faglu histofluorescence, it was found that all PI-labeled cells in SN-VTA-rr were catecholamine (CA) neurons. After a transection of the medial forebrain bundle immediately before the PI injection, an accumulation of PI was only seen in the distal segments of severed nigrostriatal CA fibers. This provides a strong evidence that PI labeling of CA cells in SN-VTA-rr is due to axonal uptake and retrograde transport.

Animals↗
PubMed1981

Propidium iodide as a nuclear marker in immunofluorescence. I. Use with tissue and cytoskeleton studies.

Some examples are given of immunofluorescence with tissue sections and microtubular cytoskeletons of cultured cells where the fluorescent dye propidium iodide (PI) has been used as marker of nuclei. The emission wave length of IP is longer than that of fluorescein, making it possible to use several different and commonly available filter combinations. The use of nuclei as positional indicators is often a more suitable method than phase microscopy combined with immunofluorescence because of low background illumination against which morphology is viewed, circumventing the need for often expensive phase optics.

Antibodies↗
PubMed1981

Propidium iodide as a nuclear marker in immunofluorescence. II. Use with cellular identification and viability studies.

The use of a fluorescence method employing propidium iodide to determine nuclear morphology of different cell types by epi-illumination in membrane immunofluorescence is described. Its usefulness is illustrated by differentiating nucleated and anuclear transferrin receptor-bearing cells as well as by simultaneously determining the viability of cell suspension without requiring a change from fluorescence microscopy, this causing no loss of visual accommodation.

Animals↗
PubMed1983

Use of propidium iodide staining and flow cytometry to measure anti-mediated cytotoxicity: resolution of complement-sensitive and resistant target cells.

Propidium iodide uptake as an indicator of cell viability was used to measure antibody-mediated cytotoxicity. In conjunction with analysis using the fluorescence-activated cell sorter, this method is as sensitive as isotope release assay systems and has the advantage of gathering multiparameter data on individual cells. This feature enables the resolution of antibody-binding target cells into a population which is sensitive to complement-mediated killing and another population which is resistant to complement-mediated killing.

Animals↗
PubMed1991

A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.

Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4 degrees C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes.

Animals↗
PubMed1996

Measurement of cytotoxicity by propidium iodide staining of target cell DNA. Application to the quantification of murine TNF-alpha.

A rapid and sensitive method is described for the determination of murine tumor necrosis factor (TNF-alpha), which can be performed in microtiter plates using a fluorescence plate scanner. The method is based on the binding of propidium iodide (PI), a membrane-impermeant dye, to nucleic acids of WEHI 164 cells, whose plasma membrane permeable due to TNF-alpha-induced cell damage. The analytical range for the proposed method is 0.3-200 pg/ml of TNF-alpha after 5 h of incubation. The optimal number of target cells was found to be 4-5X10(4)/well. The variability obtained for the PI assay was 7.6%; lower than that obtained with a commonly employed method in which MTT is used to determine cell viability (11.3%). Thus, the PI assay appears to be a reliable and reproducible method for the determination of biologically active TNF-alpha. The assay can be performed in a few hours and has the advantage over the current MTT and 51Cr-released assays that kinetic studies of TNF-alpha toxicity are possible since it permits multiple, sequenced measurements of cell viability during the incubation of the sample. The method can also be used for the determination of human TNF-alpha.

3T3 Cells↗
PubMed1984

Petite and sectored induction in Saccharomyces cerevisiae by propidium iodide: synergistic effect of sodium dodecyl sulfate.

Sodium dodecyl sulfate (SDS) was examined for its effect on petite and sectored colony induction in Saccharomyces cerevisiae by propidium iodide (PI) and ethidium bromide (EB). 4-h cultivation with 100 microM PI and 100 micrograms/ml SDS resulted in virtually all plated cells growing as sectored colonies with no decrease in viability. Sectored colonies are mixed colonies comprised of respiratory deficient and competent cells believed to be derived from an unstable respiratory deficient cell. Further cultivation with PI and SDS prior to plating led to induction of complete petite colonies with a rapid decrease in viable cells. PI alone at this concentration exhibited weak induction of sectored colonies (maximum 12.3% at 8 h) and petite colonies (maximum 10.8% at 12 h), but SDS alone caused induction of neither. 50 microM PI had almost the same activity as 100 microM except for a delay in the induction of sectored colonies in the initial stage, and a decreased rate of petite colony induction. The effects of 20 microM PI and SDS were much lower than that by 50 microM and no inhibition of growth was observed. 10 microM PI was quite inactive even in the presence of SDS. Under resting conditions, 10 approximately 100 microM PI and 100 micrograms/ml SDS induced about 60% sectored colonies at 12 h incubation and more than 60% petite colonies at 24 h. After 6 h incubation, decrease in survival was also observed.

Antimycin A↗
PubMed1990

Progressive incorporation of propidium iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity.

We describe a visual assay of neuronal electrophysiologic status for use with cultured neurons, based on the exclusion of propidium iodide (PI) by intact cellular membranes. We use this fluorescent dye, which binds to nucleic acids, at concentrations suitable for long-term exposure to neurons without toxicity. We correlate the progressive loss of resting membrane potential and the progressive inability to generate stimulated action potentials by cultured mouse dorsal root ganglion neurons with increasing incorporation of PI. The scoring system used to gauge incorporation of PI is rapid and highly reproducible using a standard fluorescence microscope. Applications exist for studies of neuronal toxicity, survival, and electrophysiology in vitro.

Animals↗
PubMed1995

Use of BCECF and propidium iodide to assess membrane integrity of acutely isolated CA1 neurons from rat hippocampus.

We used 2 fluorescent dyes, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and propidium iodide (PI), to assess the membrane integrity of neurons acutely isolated from the CA1 region of the rat hippocampus. Exciting BCECF at a relatively pH-insensitive wavelength (440 nm), or exciting PI at 490 nm, we quantitatively recorded, in real time and in single cells, the rate constants for BCECF loss (-k440) and PI uptake (k490). We found that approximately 98% of intracellular BCECF is rapidly released by applying 0.01% saponin. In neurons not treated with saponin, rate constants for BCECF loss and PI uptake typically were 1% min-1 or less under control conditions, in the presence of NH3/NH4+ and in the absence of Na+. However, in a small number of neurons, the rate constant for BCECF loss increased markedly (-k440 > 5% min-1), while pHi approached pHo, suggesting that the plasma membrane spontaneously became leaky. When neurons were progressively swollen in hypotonic solutions, rates constants for BCECF loss and PI uptake generally were affected minimally unless osmolality was decreased to approximately 75 mOsmol/kg. Treating neurons with 0.001% saponin caused an increase in PI uptake rate only in a minority of neurons, whereas in most experiments a similar treatment caused -k440 for BCECF to exceed 5% min-1, and led to a rapid deterioration of the pH gradient across the cell membrane. At even lower saponin levels (0.0005-0.0007%), we observed a much slower deterioration of pHi, which occurred at low rates of BCECF loss (-k440 = approximately 3% min-1). We conclude that computing rate constants for BCECF loss and PI uptake may be useful for assessing neuronal health, and that BCECF loss may be more sensitive to cell damage than PI uptake.

Ammonia↗
PubMed1979

Retrograde transport of bisbenzimide and propidium iodide through axons to their parent cell bodies.

Two fluorescent substances bisbenzimide (Bb), which fluoresces yellow-green and propidium iodide (PI), which fluoresces orange were found to be transported retrogradely through axons to their parent cell bodies in rat and cat. Bb gives a very strong and long lasting fluorescent retrograde neuronal labeling and is very effectively transported over long distances both in rat and cat. Bb and PI also label glial nuclei around retrogradely labeled neurons. Bb in addition labels glial nuclei along axons through which it is transported. Bb and PI can be transported retrogradely through two divergent collaterals to one and the same cell.

Animals↗
PubMed1995

Thermodynamic investigation of the association of ethidium, propidium and bis-ethidium to DNA hairpins.

We have used a combination of calorimetric and spectroscopic techniques to investigate the association of the bis-intercalator ethidium homodimer (bis-ethidium) to short DNA hairpins with sequences: d(GCGCT5GCGC) and d(CGCGT5CGCG). The helix-coil transition of each hairpin, investigated by UV and calorimetric melting protocol, takes place in monomolecular two-state transitions with characteristic enthalpies of approximately 37 kcal mol-1 for disrupting the four dG-dC base pairs of the hairpin stems. Deconvolution of the bis-ethidium-hairpin calorimetric titration curves indicate that each hairpin contains two distinct binding sites for the ligand: a high affinity site in the stem (Kb approximately 10(7)) that accommodates one bis-ethidium molecule and a lower affinity site (Kb approximately 10(6)) located probably at the loop that accommodates two bis-ethidium molecules. The overall stoichiometries of three ligands per hairpin are in agreement with those obtained in continuous variation experiments using visible spectroscopy. The interaction of bis-ethidium for each type of sites results in enthalpy driven reactions, with average binding enthalpies, delta Hb, of -13.1 and -12.1 kcal mol-1 for the stem and loop sites, respectively. Comparison to the thermodynamic profiles of ethidium and propidium binding reveals that the bis-ethidium binding to the stem site of each hairpin has a more favorable free energy term of -1.4 kcal mol-1 and more favorable enthalpy of -4.2 kcal mol-1. These suggest that only one phenanthridine ring of bis-ethidium intercalates in the stem, while the second planar ring is exposed to solvent or weakly associated to the surface of DNA.

Base Sequence↗
PubMed1996

Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells.

The effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10-100 micrograms ml-1 PI at different conductivities (0.8-14 mS cm-1) and ionic strengths, adjusted by varying concentrations of K+, Na+, Cl- and SO4(2-). Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by single-exponential curve (relaxation time about 2 min in the absence of Ca2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the 'electroleaks' during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K+ concentration (about 0.6 kV cm-1 in K(+)-free, NaCl-containing medium and about 0.9 kV cm-1 in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K(+)-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V.

Animals↗
PubMed1998

Effects of liposome-encapsulated drugs on macrophages: comparative activity of the diamidine 4',6-diamidino-2-phenylindole and the phenanthridinium salts ethidium bromide and propidium iodide.

Liposomes can be used for the intracellular delivery of drugs into macrophages. Previously, we developed a liposome-mediated macrophage 'suicide' technique based on the intraphagocytic accumulation of the liposomally delivered bisphosphonate clodronate. Later we found that the diamidine propamidine is even more effective in this approach. In the present study it is shown that liposome-encapsulated 4', 6-diamidino-2-phenylindole (L-DAPI), another well known DNA-binding diamidine, is the most effective drug in killing liver macrophages (Kupffer cells), when intravenously administered in rat. Compared to liposome-encapsulated propamidine (L-propamidine) it showed about 10-fold more activity on a molar basis. Furthermore, L-DAPI was found to induce cell death by inducing apoptosis. The structurally strongly related phenanthridinium salts ethidium bromide (EB) and propidium iodide (PI) exert marked differences in their efficacy. Whereas liposome-encapsulated PI (L-PI) was about 5 times more active in killing macrophages than L-propamidine, liposome-encapsulated EB (L-EB) showed a strongly reduced activity (10 times less than L-PI). As is shown here, PI remains mainly encapsulated in liposomes, while substantial amounts of EB leak out of liposomes. This may very well explain the differences in in vivo activity between L-EB and L-PI.

Animals↗