Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.     

Analysis and sorting of living cells according to deoxyribonucleic acid content.

The methods for measuring the deoxyribonucleic acid content of individual mammalian cells and sorting them on the basis of this parameter have until now required fixation or other treatment which renders the cells nonviable. Using a class of bis-benzimidazole dyes, Hoechst 33258 and 33342 and a multiparameter computer-controlled cell sorter, we have been able to stain and separate living cells in the G1, S, and G2+M phases of the cell cycle and to continue their growth in tissue culture with high retention of viability (greater than 90%) and no increase in heteroploidy. The quenching of the fluorescence of the bound dye by 5-bromodeoxyuridine incorporated into cellular deoxyribonucleic acid is being used with the flow system to detect and isolate mutants in deoxyribonucleic acid metabolism spectroscopically.     

Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay

The urgent need for new drug development for African trypanosomiasis is widely recognized. This requires reliable and informative high-throughput assays. Currently, drug action is determined with a fluorimetric/colorimetric assay based on the metabolism of the dye Alamar Blue (resazurin) by live cells. However, this assay does not easily distinguish between cell death and growth arrest, or supply information about the rate at which test compounds affect these parameters. We report here an alternative fluorimetric assay, based on the interaction of propidium iodide with DNA, that allows either real-time monitoring of cell viability or the generation of EC₅₀ values at a predetermined time-point. The assay is highly sensitive and fluorescence readings easily correlate to numbers of parasites or DNA content. The EC₅₀ values were highly similar to those obtained with the standard Alamar Blue assay. The procedure lends itself readily to applications in drug development or resistance monitoring.     

Interactions between N-acetyl-p-benzoquinone imine and fluorescent calcium probes: implications for mechanistic toxicology

Intracellular free calcium ([Ca2+]i) homeostasis has been implicated as an early target in both cellular necrosis and apoptosis. In this study, we have used peripheral blood mononuclear cells (PBMC) as target cells to investigate the effects of several reactive metabolites associated with drug toxicity on [Ca2+]i in order to delineate further early events in cytotoxicity. Compounds implicated in both drug-induced necrosis (N-acetyl-p-benzoquinone imine; NAPQI) and drug hypersensitivity (sulfamethoxazole hydroxylamine; SMX-HA) were examined and their effects on [Ca2+]i compared with those of the T cell mitogen phytohemagglutinin (PHA; 1.5 micrograms/ml) and the calcium ionophore ionomycin (2.5 microM). PHA and ionomycin produced characteristic elevations in [Ca2+]i as monitored by an increase in the fluorescence of fluo-3-loaded cells. SMX-HA did not significantly affect [Ca2+]i at concentrations previously shown to be cytotoxic to PBMC (100 and 500 microM), suggesting that Ca2+ homeostasis is not an early target for SMX-HA toxicity. Addition of NAPQI (250 microM) to fluo-3-loaded cells produced a marked decrease in fluorescence which was not reversed by ionomycin. Conversely, addition of NAPQI to cells loaded with indo-1 resulted in a rapid increase in fluorescence. This effect, however, was found to be attributable to NAPQI addition per se rather than to an increase in [Ca2+]i. HPLC and fluorescence analysis of samples generated from the decomposition of NAPQI revealed the presence of several products which fluoresced intensely at the excitation/emission wavelength pairs of a number of fluorescent probes commonly used to monitor [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.     

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.     

Fluorescence monitoring of antibiotic-induced bacterial damage using flow cytometry

BACKGROUND: Conventional techniques used to assess bactericidal activities of antibodies are time-consuming; flow cytometry has been used as a rapid alternative. In this study, the membrane potential-sensitive fluorescent probes bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and Sytox Green, the redox dye cyano-2,3-ditolyl tetrazolium chloride (CTC), and the Baclite viability test kit were used to assess the effects of ceftazidime, ampicillin, and vancomycin on clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively. METHODS: Bacterial cultures were grown to early exponential phase, at which point the antibiotics were added at their breakpoint values, and incubation was allowed to continue. At timed intervals, samples were stained and flow cytometric analysis was performed on a Skatron Argus 100 arc-lamp based dual-parameter flow cytometer. RESULTS: All the dyes successfully identified antibiotic-induced damage in the three strains, although different fluorescence responses between the dyes were observed. DiBAC4(3) and Sytox Green overestimated numbers of nonviable bacteria relative to loss of viability as judged by plate counts. CTC, a measure of respiratory activity, revealed antibiotic-induced population heterogeneity illus trated by the development of several subpopulations. The "live" component of the viability kit identified two populations corresponding to viable and nonviable organisms, whereas the "dead" component only revealed single populations, the fluorescence intensity of which increased with antibiotic exposure. CONCLUSIONS: Flow cytometry provides a rapid and sensitive technique for the evaluation of the antibacterial activities of antibiotics. The use of a range of fluorophores specific for different cellular characteristics may be beneficial, bearing in mind the different fluorescence responses observed among the dyes used here.     

Cytoplasmic dye transfer between metastatic tumor cells and vascular endothelium

Metastatic colonization of a secondary organ site is initiated by the attachment of blood-borne tumor cells to organ-specific adhesion molecules expressed on the surface of microvascular endothelial cells. Using digital video imaging microscopy and fluorescence activated cell sorting techniques, we show here that highly metastatic cells (B16-F10 murine melanoma and R3230AC-MET rat mammary adenocarcinoma cells) previously labeled with the fluorescent dye BCECF begin to transfer dye to endothelial cell monolayers shortly after adhesion is established. The extent of BCECF transfer to endothelial cell monolayers is dependent upon the number of BCECF-labeled tumor cells seeded onto the endothelial cell monolayer and the time of coculture of the two cell types, as visualized by an increase in the number of BCECF-positive cells among cells stained with an endothelial cell-specific mAb. Dye transfer to BAEC monolayers proceeds with a progressive loss of fluorescence intensity in the BCECF-labeled tumor cell population with time of coculture. The transfer of dye is bidirectional and sensitive to inhibition by 1-heptanol. In contrast, poorly metastatic B16-F0 melanoma cells and non-metastatic R3230AC-LR mammary adenocarcinoma cells do not efficiently couple with vascular endothelial cells. It is inferred from these experiments and from the amounts of connexin43 mRNA expressed by tumor cells that tumor cell/endothelial cell communication is mediated by gap junctional channels and that this interaction may play a critical role in tumor cell extravasation at secondary sites.     

Evidence for probenecid-sensitive organic anion transporters on polarized thyroid cells in culture

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half-maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen-coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.     

A biosensor of S100A4 metastasis factor activation: inhibitor screening and cellular activation dynamics

S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for use in vitro and in vivo. In cells, localized activation of S100A4 at the cell periphery is observed during random migration and following stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Additionally, a screen against a library of FDA-approved drugs with the biosensor identified an array of phenothiazines as inhibitors of myosin-II associated S100A4 function. These data demonstrate the utility of the new biosensor both for drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor.     

Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes

Three pH-sensitive fluorochromes-4-methyl-umbelliferone(4MU),2, 3-dicyano-hydroquinone (DCH), and 2',7'-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF)--were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pHi, which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore. Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pHi measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.     

A platinum-based covalent viability reagent for single-cell mass cytometry

In fluorescence-based flow cytometry, cellular viability is determined with membrane-impermeable fluorescent reagents that specifically enter and label plasma membrane-compromised nonviable cells. A recent technological advance in flow cytometry uses antibodies conjugated to elemental metal isotopes, rather than to fluorophores, to allow signal detection by atomic mass spectrometry. Unhampered by the limitations of overlapping emission fluorescence, mass cytometry increases the number of parameters that can be measured in single cells. However, mass cytometry is unable to take advantage of current fluorescent viability dyes. An alternative methodology was therefore developed here in which the platinum-containing chemotherapy drug cisplatin was used to resolve live and dead cells by mass cytometry. In a 1-min incubation step, cisplatin preferentially labeled nonviable cells from both adherent and suspension cultures, resulting in a platinum signal quantifiable by mass cytometry. This protocol was compatible with established sample processing steps for intracellular cytometry. Furthermore, the live/dead ratios were comparable between mass- and fluorescence-based cytometry. Importantly, although cisplatin is a known DNA-damaging agent, a 1-min "pulse" of cisplatin did not induce observable DNA damage or apoptotic responses even within 6-h post-exposure. Cisplatin can therefore be used as a viability reagent for a wide range of mass cytometry protocols.     

Fluorescent dyes for cell viability: an application on prefixed conditions

In recent years increasing attention has been given to apoptosis for its role in pathologic, organogenetic and homeostatic phenomena. Acridine orange (AO), Hoechst 33342 (HO) and propidium iodide (PI) are among the most used fluorescent dyes used to analyse cell culture viability. In fact, they respectively show specificity for living, apoptotic and late apoptosis/necrosis states. We explored whether HO, AO and PI can be used on prefixed monolayers of three commonly used cell lines. Here we mainly describe the metachromatic effects obtained by fluorescence microscopy with double and triple dye combinations. Furthermore, we propose an easy staining method in which a balanced sequential treatment with HO, AO and PI allows identification of different viability states onto fixed cells by using a long-pass FITC filter. This method extends the spectrum of suitable applications for these dyes in fluorescence viability detection onto previously fixed (prefixed) samples.     

Cytotoxicity of sulfonamide reactive metabolites: apoptosis and selective toxicity of CD8(+) cells by the hydroxylamine of sulfamethoxazole.

Treatment with sulfonamide antibiotics in HIV-infected patients is associated with a high incidence (> 40%) of adverse drug events, including severe hypersensitivity reactions. Sulfonamide reactive metabolites have been implicated in the pathogenesis of these adverse reactions. Sulfamethoxazole hydroxylamine (SMX-HA) induces lymphocyte toxicity and suppression of proliferation in vitro; the mechanism(s) of these immunomodulatory effects remain unknown. We investigated the cytotoxicity of SMX-HA via apoptosis on human peripheral blood mononuclear cells and purified cell subpopulations in vitro. CD19(+), CD4(+), and CD8(+) cells were isolated from human peripheral blood by positive selection of cell surface molecules by magnetic bead separation. SMX-HA induced significant CD8(+) cell death (67 +/- 7%) at 100 microM SMX-HA, with only minimal CD4(+) cell death (8 +/- 4%). No significant subpopulation toxicity was shown when incubated with parent drug (SMX). Flow cytometry measuring phosphatidylserine externalization 24 h after treatment with 100 microM and 400 microM SMX-HA revealed 14.1 +/- 0.7% and 25. 6 +/- 4.2% annexin-positive cells, respectively, compared to 3.7 +/- 1.2% in control PBMCs treated with 400 microM SMX. Internucleosomal DNA fragmentation was observed in quiescent and stimulated PBMCs 48 h after incubation with SMX-HA. Our data show that CD8(+) cells are highly susceptible to the toxic effects of SMX-HA through enhanced cell death by apoptosis.     

Compare Analysis for the Nanotoxicity Effects of Different Amounts of Endocytic Iron Oxide Nanoparticles at Single Cell Level

Developing methods that evaluate the cellular uptake of magnetic nanoparticles (MNPs) and nanotoxicity effects at single-cellular level are needed. In this study, magnetophoresis combining fluorescence based cytotoxicity assay was proposed to assess the viability and the single-cellular MNPs uptake simultaneously. Malignant cells (SKHep-1, HepG2, HeLa) were incubated with 10 nm anionic iron oxide nanoparticles. Prussian blue stain was performed to visualize the distribution of magnetic nanoparticles. MTT and fluorescence based assay analyzed the cytotoxicity effects of the bulk cell population and single cell, respectively. DAPI/PI stained was applied to evaluate death mechanism. The number of intracellular MNPs was found to be strongly correlated with the cell death. Significant differences between cellular MNP uptake in living and dead cells were observed. The method could be useful for future study of the nanotoxicity induced by MNPs.     

A low-volume platform for cell-respirometric screening based on quenched-luminescence oxygen sensing

Cell viability assays represent an important technology in modern cell biology, drug discovery and biotechnology, where currently there is a high demand for simple, sensitive and cost-effective screening methods. We have developed a new methodology and associated tools for cell-based screening assays, which are based on the measurement of the rates of oxygen uptake in cells by luminescence quenching. Sealable microchamber devices matching the footprint of a standard 96-well plate were developed and used in conjunction with long-decay phosphorescent oxygen probes. These devices permit cell non-invasive, real-time monitoring of cellular respiration and a rapid, one-step, kinetic assessment of multiple samples for cell viability, drug/effector action. These assays can be carried out on conventional fluorescence plate readers, they are suitable for different types of cells, including adherent and slow-respiring cells, require small sample volumes and cell numbers, and are amenable for high throughput screening. Monitoring of as little as 300 mammalian cells in 3 microl volume has been demonstrated.     

Human duodenal spheroids for noninvasive intracellular pH measurement and quantification of regulation mechanisms under physiological conditions

Three-dimensional cell cultures (spheroids) of biopsies of human duodenum were used to develop a new noninvasive method for studying intercellular and intracellular mechanisms. Through examinations of intracellular pH regulation, high functional similarity to native tissue could be shown, as already evidenced morphologically. A special microperfusion chamber was developed to fix individual spheroids physically to a nylon net, via laminar perfusion flow through the chamber. A significant improvement over current fixation methods was shown by the increase of cell viability almost up to 100%. Viability of the spheroids was confirmed by trypan blue exclusion, by a LIVE/DEAD viability/cytotoxicity kit, and by BCECF distribution. Intracellular pH was measured by use of the pH-sensitive fluorescence dye BCECF. To investigate the intracellular pH regulation, spheroid-like vesicles were acidified by NH4Cl prepulse technique. The subsequent active intracellular pH recovery was blocked with Na+-free Krebs Henseleit (KH) solution, with amiloride KH (inhibitor of the Na+-H+-exchanger), or with H2DIDS KH (inhibitor of the HCO3(-)-Cl(-)-exchanger and Na+-HCO3(-)-cotransporter). The intracellular pH of the spheroids was 7.31 +/- 0.05. pH-backregulation after acidification was prevented by sodium-free buffer, amiloride, and H2DIDS. These experiments indicated the presence of a Na+-H+-exchanger and a Na+-HCO3(-)-cotransporter. In conclusion, the human duodenal spheroid is an excellent physiological system for in vitro studies of the human duodenum.     

Estimation of intramitochondrial pCa and pH by fura-2 and 2,7 biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) fluorescence

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the fluorescent pH indicator biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The free acid forms of both probes are retained in the matrix and their fluorescence can be used to monitor the pCa and pH, respectively, of this compartment. When fura-2 loaded rat heart myocytes are lysed with digitonin, a portion of the dye is retained in the mitochondrial fraction and its fluorescence reports the uptake and release of Ca2+ by the mitochondria. It is concluded that fura-2 and BCECF may report mitochondrial as well as cytosol parameters when the probes are used in intact cells.