A rapid cell membrane permeability test using flourescent dyes and flow cytometry

A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein—fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.Abbreviations DMA dimethyl sulfate - DMSO dimethyl sulfoxide - EB ethidium bromide - F fluorescein - FDA fluorescein diacetate - FS₂₅ concentration of test substance resulting in a F-peak left-shift of 25% from control - PBS phosphate buffered saline - SCT forward light scatter - SDS sodium dodecyl sulfate     

Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.     

Fluorometric determination of DNA and RNA in Chlamydomonas using ethidium bromide

By using the fluorescence enhancement of ethidium bromide bound to nuclei acid, a very rapid, simple and sensitive assay of DNA in the green alga Chlamydomonas has been devised. Total fluorescence (DNA + RNA) was determined by complex formation with ethidium bromide in a cell lysate made by mixing cell samples with lauroyl sarcosinate, EDTA and NaOH and incubating the mixture for 5 min at room temperature followed by neutralization. For determination of DNA the RNA was digested by incubating the cell sample in te alkaline lysis solution for 45 min at 60 degrees C followed by neutralization, and complex formation with ethidium bromide. Quenching of the fluorescence due to cellular pigments was corrected for using an internal DNA standard.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

A comparison of fluorochromes for direct viable counts by image analysis

Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4',6-diamidino-2-phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.     

Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green–yellow, yellow–orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation.     

An investigation of staining methods to determine total cell numbers and the number of respiring micro-organisms in samples obtained from the field and the laboratory

Abstract Dyes were evaluated in combination with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to enable total cell numbers and the numbers of respiring cells to be determined on the same preparation. Malachite green and 4',6-diamidino-2-phenylindole (DAPI) were unsuitable counter-stains. Cells which contained INT formazan crystals could be stained with ethidium bromide or auramine. At high concentrations of INT formazan, auramine fluorescence was reduced, although this effect was partially rectified by prior fixation with glutaraldehyde. Staining with ethidium bromide produced a strong fluorescence in cells containing crystals of INT formazan. This observation was developed into a procedure which allowed total cells to be determined and provided a useful estimate of the number of respiring cells in samples obtained from the laboratory and the field.     

Subpopulation analysis of drug-induced cell-cycle delay in human tumor cells using 90 degrees light scatter

A mitotic cell subset has been identified with nuclear light scatter. Colcemid-treated T-47D human breast cancer cells were permeabilised, stained with ethidium bromide, and analysed by flow cytometry. Cells with G2M DNA content exhibited a unimodal distribution for DNA fluorescence and forward scatter, but two peaks were discernible with 90 degrees light scatter. A discrete low-scattering cell cluster could be distinguished from the G2 cell subset on two-dimensional contour plots of 90 degrees light scatter vs. DNA fluorescence; this cluster was reproduced by mitotic shake-off experiments and varied quantitatively with mitotic indices determined either by microscopy or by stathmokinetic cell-cycle analysis of DNA fluorescence. Cell sorting confirmed that the low-scattering cell cluster comprised predominantly metaphase and anaphase cells. Identification of mitotic cells with this one-step technique enables rapid analysis of drug-induced cell-cycle delay in cell populations with different rates of cell-cycle traverse. Hence, vincristine-induced cytostasis is shown to arise in part because of premitotic G2 arrest, whereas etoposide is shown to affect cycling cells with equal sensitivity in quiescent and activated cell populations. The use of light scatter to discriminate mitotic cells in this way facilitates analysis of drug-induced cell-cycle delay and supplements the information obtainable by conventional cell-cycle analysis.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

Influence of ethidium bromide on hyperthermic modification of bleomycin-DNA interaction

The effect of hyperthermic treatment on the binding of 59Fe-labeled bleomycin to DNA has been studied. Enhanced binding was observed at elevated temperatures. The influence of the DNA-intercalating agent, ethidium bromide, on bleomycin-DNA interaction was also studied and revealed a considerable decrease in this interaction at ethidium bromide levels below 1 microgram/ml. Ethidium bromide was observed to remove the enhanced bleomycin-DNA interaction recorded previously following incubation at hyperthermic temperatures. Synergistic action of bleomycin and hyperthermia on loss of clonogenic ability of HT29R cells is reported. Incubation of cells under hyperthermic conditions with bleomycin in the presence of ethidium bromide removes this synergism, producing a less than additive effect for the action of bleomycin and heat after ethidium bromide effects are taken into account.     

Two-Step Cell-Death Kinetics In Vitro During Cis-Platinum, Hydroxyurea and Mitomycin Incubation

A human leukaemic cell line (REH) growing in suspension was incubated with cis-platinum, hydroxyurea and mitomycin C at various concentrations causing complete cell-cycle arrest. At different times the cell suspensions were harvested, diluted 1:1 with a buffer solution, stained without further treatment with a mixture of acridine orange (AO) and ethidium bromide (EB) and analysed with a biparametrical flow cytometer. Fluorescent plastic beads were introduced into the suspensions to provide an internal numerical reference for the control of cell loss. The fluorescence distributions showed three groups of cells: vital cells (V) which were only stained with AO; dead cells in which EB stained cytoplasmic components but not the nuclear DNA (D1), and dead cells which allowed EB to stain both cytoplasm and nuclear DNA (D2). the kinetics of cells entering D1 depended on drug concentration and showed equal characteristics for cis-platinum and mitomycin, but were different for hydroxyurea. the subsequent entry into D2 occurred about 15 hr later and showed no pronounced dependence on drug concentration. Parallel trypan-blue (TB) exclusion tests revealed that TB only stained D2 cells and therefore is not useful for investigating cell-death kinetics during exposure to cell-killing agents.     

Two-step cell-death kinetics in vitro during cis-platinum, hydroxyurea and mitomycin incubation

A human leukaemic cell line (REH) growing in suspension was incubated with cis-platinum, hydroxyurea and mitomycin C at various concentrations causing complete cell-cycle arrest. At different times the cell suspensions were harvested, diluted 1:1 with a buffer solution, stained without further treatment with a mixture of acridine orange (AO) and ethidium bromide (EB) and analysed with a biparametrical flow cytometer. Fluorescent plastic beads were introduced into the suspensions to provide an internal numerical reference for the control of cell loss. The fluorescence distributions showed three groups of cells: vital cells (V) which were only stained with AO; dead cells in which EB stained cytoplasmic components but not the nuclear DNA (D1), and dead cells which allowed EB to stain both cytoplasm and nuclear DNA (D2). The kinetics of cells entering D1 depended on drug concentration and showed equal characteristics for cis-platinum and mitomycin, but were different for hydroxyurea. The subsequent entry into D2 occurred about 15 hr later and showed no pronounced dependence on drug concentration. Parallel trypan-blue (TB) exclusion tests revealed that TB only stained D2 cells and therefore is not useful for investigating cell-death kinetics during exposure to cell-killing agents.     

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.     

Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting

We have used highly specific, directly fluorescein-conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10-5 to 10-6. We were able to isolate the variants by first sorting for an approximate 1000-fold enrichment of the desired immunoglobulin-producing cells, growing these cells for five to nine days, followed by a second 1000-fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3-5 weeks after the original selection for secretion of dansyl-binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, "red" fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.     

Cytological Analysis of Anastomoses and Vegetative Incompatibility Reactions in <Emphasis Type="Italic">Helicobasidium monpa</Emphasis>

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus. Received: 13 June 2001 / Accepted: 8 August 2001     

Early increase in intracellular calcium during photodynamic permeabilization

The purpose of this study was to determine if there was an early increase in intracellular Ca++ which preceded generalized lysis of thymocytes during photodynamic permeabilization. A method was developed that facilitated the simultaneous measurement in real time of permeabilization of the thymocyte cell membrane to Ca++, Mn++, and ethidium bromide during photodynamic action. Quin-2 loaded cells were illuminated in the presence of erythrosin B and the change in the fluorescence emission of the calcium-quin-2 complex was used to determine how soon and to what extent intracellular Ca++ changed following illumination. In the presence of extracellular manganese, the same system was used to determine how soon the cells became permeable to Mn++ or quin-2. It was determined that the fluorescence emission of the ethidium bromide-DNA complex was strong enough to be measured in the presence of the calcium-quin-2 complex. This enabled the concomitant determination of the elapsed time following illumination before ethidium bromide entered the cell. It was established that increased intracellular Ca++ was an early event in the photodynamic permeabilization of thymocytes that preceded permeabilization of the cell membrane to ethidium bromide, Mn++ or quin-2, or lysis.     

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.     

Fiuorimetric assessment of Pseudomonas fluorescens viability after freeze-thawing using ethidium bromide

E.O. PUCHKOV AND A.N. MELKOZERNOV. 1995. The relationship between impairment of the Pseudomonas fluorescens cell envelope's permeability barrier for ethidium cation, the fluorescent moiety of ethidium bromide, and viability after freeze-thawing was investigated. Ethidium fluorescence in the suspension of intact bacteria did not change. Disruption of the bacterial permeability barrier by cetyltrimethylammonium bromide (CTAB) led to ethidium fluorescence increase due to interaction of the fluorochrome with intracellular nucleic acids. In the suspension of freeze-thawed cells, ethidium fluorescence increased and the subsequent treatment by CTAB resulted in further fluorescence increase up to the final level corresponding to that in CTAB-treated intact bacteria. For bacteria exposed to different freeze-thawing regimes, the relative ethidium fluorescence increase closely correlated with the relative number of fluorescing cells revealed microscopically. In the suspension of freeze-thawed cells, the relative additional ethidium fluorescence increase after CTAB treatment closely correlated with viability evaluated by plate counts. It is concluded that the fluorimetric approach may be used as a means of rapidly evaluating bacterial viability after freeze-thawing.     

Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells.

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.     

A test of granulocyte membrane integrity and phagocytic function.

An assay of granulocyte viability has been developed which yields information about rwo important cell parameters, cell membrane integrity and phagocytic activity. The assay is based on the fact that only live cells can accumulate fluorescein, which is enzymatically split from the nonfluorescent substrate fluorescein diacetate. Dead cells, on the other hand, become permeable to the fluorescent red dye ethidium bromide. When cells are exposed first to opsonized zymosan particles, which they can phagocytize, then to a combination of these fluorescent dyes, one can distinguish microscopically between dead cells with fluorescent red nuclei, live cells which fluoresce green, and live cells with phagocytic function which are swollen with the pink zymosan particles in a green fluorescing cytoplasm. This assay takes 20--30 min and can be used to distinguish different degrees of cellular damage after cryopreservation.