Perchlorotrityl radical-fluorophore conjugates as dual fluorescence and EPR probes for superoxide radical anion

Perchlorotrityl radicals, mono-substituted with a fluorophore using an amide linker of varying chain length, were synthesized and characterized. Electron paramagnetic resonance (EPR) spectroscopic study indicated free-electron coupling with the aromatic hydrogen nuclei and long-range coupling with the methylene hydrogens of the linker group. Reactivity of the fluorophore-conjugated trityls with superoxide radical anion showed quenching of EPR signal and enhancement of fluorescence emission spectrum. This work presents the first example of a perchlorotrityl-fluorophore conjugate that can potentially be employed as a dual probe for the detection of superoxide under oxidative stress-mediated conditions in biological systems.     

Development of a Novel Fluorophore for Real-Time Biomonitoring System

Rapid in-field diagnosis is very important to prevent the outbreak of various infectious and contagious diseases. Highly sensitive and quantitative detection of diseases can be performed using fluorescent immunochemical assay with specific antigen-antibody binding and a good quality fluorophore. This can lead to the development of a small, portable, quantitative biosensor to transmit diagnostic results to a control center in order to systematically prevent disease outbreaks. In this study, we developed a novel fluorophore, coumarin-derived dendrimer, with high emission intensity, strong signal brightness, and high photostability. It is easily coupled with biomolecules and emits strong and stable fluorescence at 590 nm with excitation at 455 nm. Application to fluorescent immunochromatographic test (FICT) showed that the novel coumarin-derived dendrimer bioconjugate could detect antigens at amount as low as 0.1 ng. The clinical results and the spectral characteristics of the novel coumarin-derived dendrimer open, for the first time, the possibility of developing a cost/energy efficient LED-based portable quantitative biosensor for point-of-care (POC) disease diagnosis, which can permit real time monitoring (U-healthcare system) by a disease control center.     

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.     

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.     

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.     

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.     

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.     

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements.

BACKGROUND: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements. METHODS: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and beta2-microglobulin with antibodies conjugated with fluorescein- and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray. RESULTS: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye- and instrument-dependent parameters were calculated for all matching pairs of seven fluorophores on the three commercial cytometers. The most crucial parameter in determining the applicability of the donor-acceptor pairs was the normalized fluorescence intensity and the least important one was the spectral overlap. CONCLUSIONS: On the basis of available laser lines, the optimal dye pair for all three cytometers is the Alexa546-Alexa647 pair, which produces high energy transfer efficiency values and has the best spectral characteristics with regard to laser excitation, detection of emission, and spectral overlap.     

The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer

A simple and sensitive fluorescence anisotropy method was developed for lysozyme, employing the coupling of fluorophore, 6-carboxyfluorescein (FAM), with lysozyme upon recognition between the target molecule and its DNA aptamer. It was found in this study that the rotational dynamic of the detecting system is crucial to obtain a high anisotropy signal that cannot always be achieved by simply increasing the molecular volume, because molecular volume increase may not be able to efficiently retard the rotational movement of the fluorophore. FAM was selected as the label of the ssDNA aptamer to effectively facilitate the change of the fluorophore from a primarily independent segmental movement to slow global rotation. The time-resolved measurements, including lifetime and dynamic fluorescence anisotropy, were conducted to study the recognition interaction and to better understand the methodology. The proposed method had a wide linear dynamic range of 12.5-300 nM and a high sensitivity with the limit of detection of 4.9 nM (3S/N). This proposed method was successfully applied to assay of human salivary lysozyme. The results based on the standard addition recovery and comparison with enzyme-linked immunosorbent assay (ELISA) demonstrated the feasibility of this method for biological samples. Using coupling between the fluorophore and the analyte can be one of the approaches working toward expanding the application of fluorescence anisotropy based on aptamer-target and antibody-antigen recognitions.     

Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin

In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.     

Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.     

A DIE responsive NIR-fluorescent cell membrane probe

It is challenging to achieve selective off to on modulation of the emissive state of a fluorophore within a complex and heterogeneous cellular environment. Herein we show that the dis-assembly of a non-fluorescent aggregate to produce individual fluorescent molecules, termed disaggregation induced emission (DIE), can be utilised to achieve this goal with an amphiphilic BF₂-azadipyrromethene (NIR-AZA) probe. Optical near-infrared properties of the NIR-AZA probe used in this study include absorption and emission maxima at 700 and 726 nm respectively when in the emissive non-aggregated state. Key to the success of the probe is the bis-sulfonic acid substitution of the NIR-AZA fluorophore, which is atypical for membrane probes as it does not contain zwitterionic lipid substituents. The aggregation/disaggregation properties of the NIR-fluorophore have been investigated in model surfactant and synthetic liposomal systems and shown to be emissive responsive to both. Real-time live cell imaging experiments in HeLa Kyoto and MC3T3 cells showed a rapid switch on of emission specific to the plasma membrane of viable and apoptotic cells attributable to a disaggregation-induced emission of the probe. Image analysis software confirmed localisation of fluorescence to the plasma membrane. Cell membrane staining was also effective for formaldehyde fixed cells, with staining possible either before or after fixation. This study adds new and important findings to recent developments of DIE responsive probes and further applications of this controllable emission-switching event are anticipated.     

Intracellular ribozyme-catalyzed trans-cleavage of RNA monitored by fluorescence resonance energy transfer

Small catalytic RNAs like the hairpin ribozyme are proving to be useful intracellular tools; however, most attempts to demonstrate trans-cleavage of RNA by ribozymes in cells have been frustrated by rapid cellular degradation of the cleavage products. Here, we describe a fluorescence resonance energy transfer (FRET) assay that directly monitors cleavage of target RNA in tissue-culture cells. An oligoribonucleotide substrate was modified to inhibit cellular ribonuclease degradation without interfering with ribozyme cleavage, and donor (fluorescein) and acceptor (tetramethylrhodamine) fluorophores were introduced at positions flanking the cleavage site. In simple buffers, the intact substrate produces a strong FRET signal that is lost upon cleavage, resulting in a red-to-green shift in dominant fluorescence emission. Hairpin ribozyme and fluorescent substrate were microinjected into murine fibroblasts under conditions in which substrate cleavage can occur only inside the cell. A strong FRET signal was observed by fluorescence microscopy when substrate was injected, but rapid decay of the FRET signal occurred when an active, cognate ribozyme was introduced with the substrate. No acceleration in cleavage rates was observed in control experiments utilizing a noncleavable substrate, inactive ribozyme, or an active ribozyme with altered substrate specificity. Subsequently, the fluorescent substrates were injected into clonal cell lines that expressed cognate or noncognate ribozymes. A decrease in FRET signal was observed only when substrate was microinjected into cells expressing its cognate ribozyme. These results demonstrate trans-cleavage of RNA within mammalian cells, and provide an experimental basis for quantitative analysis of ribozyme activity and specificity within the cell.     

Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay

The oxidation of 2′,7′-dichlorodihydrofluorescein (2′,7′-dichlorofluorescin, DCFH) to a fluorescent product, 2′,7′-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates.     

Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay

The oxidation of 2',7'-dichlorodihydrofluorescein (2',7'-dichlorofluorescin, DCFH) to a fluorescent product, 2',7'-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates.     

A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors

Phosphorylation of extracellular signal-regulated kinase (ERK) is linked to activation of many cell surface receptors and kinases. However, phosphorylated ERK has not been used as a biochemical marker to monitor pharmacology of these biomolecules, largely because commonly used methods to detect the phosphoprotein are not quantitative and do not have sufficient throughput. In this article, a high-throughput, 384-well, cell-based functional assay to quantify dopamine agonist-induced ERK phosphorylation in D2- and D3-overexpressed cell lines is described. The assay uses infrared-labeled secondary antibodies to detect phospho-ERK, and the signals in the wells of the microtiter plate are quantified by a LI-COR infrared scanner. V(max), EC(50), and functional K(i) values of various D2 and D3 agonists and antagonists determined in this assay are similar to those in the literature. The assay is nonradioactive, is quantitative, and has a good signal-to-noise ratio. In addition, the signal is stable. This assay can be used to monitor the activities of many G protein-coupled receptors and other signaling biomolecules that are linked to phosphorylation of ERK. The methodology can potentially be used to detect the change in level of any cellular protein in which highly selective antibodies are available.     

Detection by the comet assay of apoptosis induced in lymphoid cell lines after growth factor deprivation

Dysregulation of apoptosis contributes to various diseases such as neurodegenerative or aging disorders, autoimmune syndromes or cancers. Numerous experimental paradigms have been explored to characterize molecular and cellular modulators of apoptosis. Similarly, numerous techniques have been described for detecting and/or quantifying accurately cells committed to apoptosis. Besides the conventional techniques, we describe in this report that the comet assay, which detects DNA single- and double-strand breaks in situ, at the cellular level, is relevant for the characterization of apoptotic cells. The comet assay is very sensitive and detects DNA fragmentation occurring in the apoptotic process as early as exposure of phosphatidylserine residues on the outer leaflet. Thus the comet assay can be used for the recognition of apoptosis that follows the death signal caused, for example, by genotoxic stress as well as lack of survival signal as in growth factor deprivation.     

A comparison of the emission efficiency of four common green fluorescence dyes after internalization into cancer cells

In vivo optical imaging to enhance the detection of cancer during endoscopy or surgery requires a targeted fluorescent probe with high emission efficiency and high signal-to-background ratio. One strategy to accurately detect cancers is to have the fluorophore internalize within the cancer cells permitting nonbound fluorophores to be washed away or absorbed. The choice of fluorophores for this task must be carefully considered. For depth of penetration, near-infrared probes are ordinarily preferred but suffer from relatively low quantum efficiency. Although green fluorescent protein has been widely used to image tumors on internal organs in mice, green fluorescent probes are better suited for imaging the superficial tissues because of the short penetration distance of green light in tissue and the highly efficient production of signal. While the fluorescence properties of green fluorophores are well-known in vitro, less attention has been paid to their fluorescence once they are internalized within cells. In this study, the emission efficiency after cellular internalization of four common green fluorophores conjugated to avidin (Av-fluorescein, Av-Oregon green, Av-BODIPY-FL, and Av-rhodamine green) were compared after each conjugate was incubated with SHIN3 ovarian cancer cells. Using the lectin binding receptor system, the avidin-fluorophore conjugates were endocytosed, and their fluorescence was evaluated with fluorescence microscopy and flow cytometry. While fluorescein demonstrated the highest signal outside the cell, among the four fluorophores, internalized Av-rhodamine green emitted the most light from SHIN3 ovarian cancer cells both in vitro and in vivo. The internalized Av-rhodamine green complex appeared to localize to the endoplasmic vesicles. Thus, among the four common green fluorescent dyes, rhodamine green is the brightest green fluorescence probe after cellular internalization. This information could have implications for the design of tumor-targeted fluorescent probes that rely on cellular internalization for cancer detection.     

Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain

We have developed a simple, sensitive, fluorescence microplate-based assay for tumor necrosis factor (TNF) biological activity. The assay employs SYTOX Green nucleic acid stain to detect TNF-induced cell necrosis in actinomycin D sensitized cultured cell lines. SYTOX Green stain is a cationic unsymmetrical cyanine dye that is excluded from live cells but can readily penetrate cells with compromised cell membranes. Upon binding to cellular nucleic acids, the dye exhibits a large enhancement in fluorescence, which is monitored at fluorescein wavelengths. We detected 2.5 pg/mL and quantitated 25-500 pg/mL recombinant murine (rm) and recombinant human (rh) TNF-alpha, using mouse fibroblast-derived WEHI 164, WEHI 13var, and L929 cell lines. The procedure can also be used to detect agents that modulate TNF activity. We demonstrated complete inhibition of rhTNF-alpha using monoclonal anti-human TNF-alpha antibody and determined that approximately 20 ng/mL antibody was sufficient to neutralize 50% of the biological activity of 250 pg/mL rhTNF-alpha in these cell lines. Reagents are added in a single step, followed by a 6- to 8-h incubation period, during which the cytokine exhibits its effects. There are no wash steps, and the assay is readily amenable to automation and high-throughput screening procedures.