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The fluorescence intensity of propidium iodide bound to DNA depends on the concentration of sodium chloride.

While performing DNA analysis with propidium iodide using the FACS-II cell sorter, it was noted that the fluorescence intensity was not constant during the measurements. When different sheath and sample fluids were used, the fluorescence intensity of a given cell population increased or decreased during the measurement, depending on the concentration of sodium chloride in the fluids. By selecting the appropriate sheath and sample fluid combination, the drift in fluorescence intensity can be avoided.

Animals↗

Comparison of two flow cytometric assays for cellular RNA--acridine orange and propidium iodide.

Two flow cytometric assays for cellular RNA, two-step acridine orange (TSAO) and propidium iodide (PI), were compared with each other and with ultraviolet (uv) spectrophotometry of RNA to determine their ability to quantitate cellular RNA and to differentiate between proliferating and quiescent (Q) cells. The model system used for these comparisons was cells from unfed cultures of two mouse mammary tumor lines designated 66 and 67. The growth kinetics of unfed 67 and 66 cells were characterized by a decrease in cellular RNa (a factor of 2) with the decrease occurring throughout the entire population of cells as they entered plateau phase. The time course of the RNA decrease as monitored by the PI assay closely corresponded to that observed by uv spectrophotometric measurements. The TSAO assay, however, agreed with the uv spectroscopy and PI assays on the extent of the RNA decrease but showed no decrease in RNA until 24 hours after the other assays indicated that a significant decrease had occurred. The plateau cells from 67 and 66 unfed cultures had a greatly lowered RNA content and were predominately (greater than 97%) in the G1 phase of the cell cycle in terms of DNA content. However, when compared with exponentially growing cells, they were a distinct Q population. This distinction could be observed using either the TSAO or the PI assay. The TSAO assay provided better resolution of the Q population and has the added advantage of giving DNA distribution simultaneously. Therefore, both flow cytometric assays are particularly useful in this system; the TSAO for monitoring the Q population and the PI for determination of kinetic changes in total cellular RNA.

Acridine Orange↗

Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence.

The relative ease with which a flow cytometer can perform simultaneous two color immunofluorescence to examine subpopulations of lymphoid cells has been well documented. Thus, flow cytometers equipped with only a single argon laser can be used to delineate various cell types by exciting both fluorescein- and phycoerythrin-conjugated antibodies to cell surface antigens. One problem that remains, however, is the artifactual staining of dead cells and clumps, which cannot be distinguished from viable cells on the basis of cell surface staining characteristics. We describe a method for simultaneous two color analysis or sorting of viable leukocytes which requires only a single laser. The method utilizes propidium iodide, which stains dead cells and thereby excludes such cells from the analysis. Using this method, as many as four viable cell types have been simultaneously analyzed in a single sample.

Cell Line↗

Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5-bromo-2'-deoxyuridine and propidium iodide.

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.

Animals↗

Measurement of S-phase fractions in lymphoid tissue comparing fresh versus paraffin-embedded tissue and 4',6'-diamidino-2 phenylindole dihydrochloride versus propidium iodide staining.

S-phase fractions for 62 lymphoid biopsies were calculated, by means of flow cytometry, from both fresh and paraffin-embedded tissue. The purposes of this study were to determine whether significant differences were seen between S-phase estimates obtained from fresh and fixed tissue and to compare results obtained with two DNA dyes, namely 4'-6'-diamidino-2 phenylindole dihydrochloride (DAPI) and propidium iodide (PI). The 62 cases consisted of 38 cases of non-Hodgkin's lymphoma (NHL), 19 reactive samples, and 5 cases of Hodgkin's disease. Fifty-four of the samples showed DNA diploid profiles. A good agreement between S-phase results from fresh and fixed tissue was seen, with technical factors accounting for around 20% of the total variance. Using a paired t test, no significant difference was seen between fresh and fixed tissue for diploid cases, but there was a trend for S-phase estimates from fixed tissue to be higher. If all cases (including the eight DNA aneuploid samples) were included in the analysis this difference just reached statistical significance (P less than .05). In a subgroup of 19 of the cases, a comparison was performed on both fresh and fixed tissue of staining with DAPI and PI. A good agreement between results with both DNA stains was found on fresh and fixed tissue, with no significant differences being apparent, and stain-related factors accounted for only 10% of the total variance.

Cell Cycle↗

Flow cytometric evaluation of DNA stainability with propidium iodide after histone H1 extraction.

A flow cytometric evaluation of the effect of the histone H1 extraction on DNA stainability with propidium iodide was performed on isolated HeLa nuclei. Selective removal of the lysine-rich protein was attained by using two established techniques involving treatment with 0.7 M NaCl or low pH. DNA stainability was monitored at different dye/DNA-P ratios, varying from low to high saturating concentrations. Depletion of the histone H1from nuclei results in the transition from low to high affinity of a portion of binding sites, as shown by 1) the increase in fluorescence intensity after staining with the dye at low saturating concentrations and 2) the higher value of the fluorescence intensity ratio (FI5/FI50) exhibited by H1-depleted nuclei stained with a low (5 micrograms/ml) vs. a high (50 micrograms/ml) concentration, as compared with control samples.

Cell Nucleus↗

Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide.

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strand-specific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enzymes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nuclease-induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios greater than 0.11. Below this value (2 micrograms/ml PI), the fluorescence intensity of digested samples was 15-30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation- but not sequence-specific nucleases induce a relaxation of DNA supercoils.

Coloring Agents↗

Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst-propidium iodide stain.

Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis-benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd-Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics.

Animals↗

Fluorescence instability of propidium iodide-labelled microspheres in (para)formaldehyde.

An instability of the fluorescence of propidium iodide (PI)-labelled microspheres was observed when the beads were used as an internal fluorescence standard in cell samples suspended in paraformaldehyde (pFA) for flow cytometric phenotyping. Flow cytometry of fluorochrome-labelled microspheres as well as spectrofluorometry of PI-fluorochrome solutions in PBS-buffered 1% pFA or formalin (FA) revealed strong increases of PI fluorescence starting immediately after the addition to the fixatives. We propose a chemical reaction which leads to two additional conjugated double bonds in the modified fluorochrome causing the increased fluorescence emission. Therefore, PI-labelled microspheres should not be applied as internal fluorescence references in aldehyde-containing cell suspensions for flow cytometric analysis, except when the time interval between the addition of beads and the measurement can be kept constant or if it lasts longer than 1 h.

Flow Cytometry↗

Propidium iodide and the thiol-specific reagent DACM as a dye pair for fluorescence resonance energy transfer analysis: an application to mouse sperm chromatin.

The dyes N-(-7-dimethyl-amino-4-methyl-coumarinyl) maleimide and propidium iodide, specific for the thiol group and DNA, respectively, were considered as a donor-acceptor couple suitable for investigating "in situ" the relative spatial distribution of DNA and protamines in mouse spermatozoa chromatin. The two dyes are characterized by favourable spectral properties, so that a simplified analytical procedure, based on the measurement of both donor and acceptor emission in double-stained samples, can be applied to evaluate the relative efficiency of the energy transfer process and its topological distribution. The results obtained indicate that during the maturation process: 1) the basic arrangement of protamine-DNA complex does not undergo structure changes, and 2) the oxidation of sulfhydryl to disulfide groups, resulting in chromatin stabilization, first involves the protamine thiols spatially closer to DNA. Fluorescence energy transfer imaging suggests that chromatin stabilization starts in the midportion of the sperm head, then spreads towards the periphery.

Animals↗

Simultaneous measurement of two cellular antigens and DNA using fluorescein-isothiocyanate, R-phycoerythrin, and propidium iodide on a standard FACScan.

Multiparameter flow cytometry is a powerful tool for analyzing the phenotypic, cell kinetic, and ploidy heterogeneity of tumor cell populations. Because of the substantial spectral overlap of propidium iodide (PI) and R-phycoerythrin (PE) fluorescence emission, this combined use of these fluorochromes has been thought not to be feasible on a standard flow cytometer for these kind of studies. Instead of PI, 7-amino-actinomycin D (7-AAD) is used as DNA stain. In this paper however, we show that PI can be used as a DNA stain in combination with fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) on a standard FACScan. Three established ovarian cancer cell lines (IGROV1, NIH: OVCAR-3, and COV362.c14) were used for these experiments. Cells were fixed with 1.0% paraformaldehyde and permeabilized with various concentrations of lysolecithin for the simultaneous detection of surface antigens by monoclonal antibodies MOv18, BMA180 or OV632, intermediate filament antigens (keratin 18 or vimentin), and DNA. A final concentration of 80 micrograms/ml lysolecithin was found to give optimal results. The emission spectrum overlap from PI into the orange fluorescence channel (FL2) used for PE fluorescence detection could be sufficiently compensated up to a photomultiplier tube potential of about 440 Volts (V) required at the FL2 channel. Using the same instrument settings, 5.10 x 10(4) PE equivalents were detectable. Under these conditions, CVs obtained for the DNA histograms ranged from 3.0-4.1. Application of the method on a mixture of activated peripheral blood lymphocytes and ovarian tumor cells resulted in a clear separation of the two populations both by surface and cytoplasmic antigen expression and DNA content.

Antibodies, Monoclonal↗

Reversing the effect of formalin on the binding of propidium iodide to DNA.

Formalin, an excellent preservative of cellular morphology, is a commonly used fixative for tissue specimens in hospital pathology laboratories. This preserved material is a potential source of tissue for diagnostic and retrospective research studies on DNA using flow cytometry. Unfortunately, formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA, thus altering the measurement of DNA content by flow cytometry or image analysis. This interference has been attributed to the cross-linking of histones by formalin. Since formalin alters the measurement of DNA content in formalin-fixed and formalin-fixed, paraffin-embedded tissues, this study was designed to explore the use of various physicochemical methods to reverse the effect of the formalin on the binding of PI to DNA. This study demonstrates that resuspending formalin-fixed cells in PBS and heating them at 75 degrees C for at least 1 h prior to staining with PI restores the staining of the DNA to approximately the same fluorescence intensity as that of fresh tissue.

Artifacts↗

A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells.

A flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensation with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it was possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apoptotic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured polymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cell sorting of the presumed apoptotic population, followed by morphologic examination or DNA analysis of the sorted populations. The usefulness of such a method is discussed in terms of applications in the analysis of heterogeneous clinical samples, populations with low DNA degradation during apoptosis, and cell cycle position of the apoptotic cells.

Antineoplastic Agents↗

TO-PRO-3 iodide: a novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry.

A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper. A subequilibrium concentration of 1 microM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In human- and mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation. Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously.

Animals↗

An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry.

Previously, we described GFP-spectrin, a membrane-localized derivative of the green fluorescent protein that can be employed as a marker during the simultaneous identification of transfected cells and cell cycle analysis by flow cytometry (Kalejta et al., Cytometry 29: 286-291, 1997). A membrane-anchored GFP fusion protein is necessary because the ethanol permeabilization step required to achieve efficient propidium iodide staining allows cytoplasmic GFP to leach out of the cell. However, viable cells expressing GFP-spectrin are not as bright as cells expressing cytoplasmic GFP and their fluorescence intensity is further diminished after ethanol treatment. Here, we demonstrate that the fluorescence intensity of cells expressing an integral membrane GFP fusion protein (Us9-GFP) is similar to that of cells expressing cytoplasmic GFP and is quantitatively maintained in cells after ethanol treatment. By allowing an accurate assessment of the expression level of GFP, Us9-GFP allows a more precise analysis of the effects of a cotransfected plasmid on the cell cycle and thus represents an improvement upon the original membrane-associated GFP fusion proteins employed in this assay.

Cell Cycle↗

Neural substrate of a cerebellar movement disorder induced by intracerebroventricular injection of propidium iodide in the rat: a Fos immunocytochemical study.

Intracerebroventricular (icv) injection of propidium iodide (PI) in the rat results in a transient movement disorder characterized by nystagmus, ataxia, and shaking. In the present study we used c-Fos as a marker for neuronal activation to investigate the neural substrate underlying this movement disorder. PI was injected into the lateral cerebral ventricle of freely moving rats through a previously implanted cannula. Animals were perfused 3 h after the injection and the brains were processed for c-Fos immunocytochemistry. Paired control animals were injected with saline. After PI injection, a significant Fos expression was seen in the cerebral cortex, thalamic midline nuclei, thalamic intralaminar nuclei, hypothalamus, central gray, pontine nuclei, locus coeruleus, vestibular complex, inferior olive, ventrolateral medulla, nucleus of solitary tract, and deep cerebellar nuclei. Few or no Fos immunoreactive cells were seen in the above structures of the control animals. The present study indicates that a large number of neurons located in many different neural structures are activated following icv injection of PI. Second, consistent with the cerebellar feature of the movement disorder, a major Fos expression was found in the cerebellar circuitry (deep cerebellar nuclei, pontine nuclei, vestibular complex, and inferior olive). It reinforces further the assumption that the movement disorder is due to cerebellar dysfunction caused by PI.

Animals↗

Infarct size measurement by triphenyltetrazolium chloride staining versus in vivo injection of propidium iodide.

Infarct size delineation by triphenyltetrazolium chloride (TTC) staining is dependent on sufficient reperfusion. We therefore evaluated the possibility of using propidium iodide (PI), a reagent conventionally used in flow cytometry to fluorescently stain dead cells, for infarct size analysis after short periods of reperfusion. Forty-five rabbits were subjected to either 15 min, 2 h or 4.5 h of coronary artery occlusion without reperfusion, or to 15 min, 30 min and 2 h of coronary artery occlusion followed by 30 min, 1 h and 3 h of reperfusion. Fifteen min before terminating the experiment, PI was injected into the left atrium. Patent blue violet was used to delineate the area at risk. Following incubation in TTC, the area at risk was excised and cross sections obtained for microscopical infarct size quantification by PI fluorescence. PI fluorescence was absent after permanent occlusion and in control areas. Infarct sizes measured by TTC staining were significantly smaller after 1 h of reperfusion as compared to 3 h of reperfusion (30 min occlusion: 1+/-1 v 34+/-9%; P<0.05; 2 h occlusion: 9+/-6 v 47+/-8%; P<0.01). In contrast, infarct sizes determined by PI fluorescence reached values comparable to those measured by TTC staining or conventional histology after longer times of reperfusion already after 30 min of reperfusion (30 min occlusion: 35+/-16.5%; 2 h of occlusion: 61+/-8%). Therefore, after short times of reperfusion infarct size measurement by PI fluorescence is more reliable than by TTC staining.

Animals↗

Propidium iodide staining of cytoautoradiographic preparations for the simultaneous determination of DNA content and grain count.

A new method is described for staining cell nuclei with propidium iodide in preparations that have been processed for autoradiography. The permeability of the stripping film to the dye molecule has been studied, as have the staining conditions, in order to optimize the stoichiometry of the DNA-dye interaction. A procedure has also been set up to allow the simultaneous measurement of DNA content and grain count on the same cell.

Anemia, Hemolytic↗