SYTOX Green Nucleic Acid Stain Research Articles

The FcγRIII Engagement Augments PMA-Stimulated Neutrophil Extracellular Traps (NETs) Formation by Granulocytes Partially via Cross-Talk between Syk-ERK-NF-κB and PKC-ROS Signaling Pathways.

Polymorphonuclear neutrophils (PMNs) are the most abundant white blood cell in the circulation capable of neutrophil extracellular traps (NETs) formation after stimulation. Both NADPH oxidase-dependent and -independent pathways are involved in NET formation. The IgG is the most abundant immunoglobulin in human serum. However, the impact of the circulating IgG on NET formation is totally unexplored. In this study, the all-trans retinoic acid (ATRA)-induced mature granulocytes (dHL-60) were pre-treated with monomeric human IgG, papain-digested Fab fragment, crystallizable IgG Fc portion, rituximab (a human IgG1), or IgG2. The NET formation of the dHL-60 in the presence/absence of phorbol 12-myristate 13-acetate (PMA) stimulation was then measured by the fluorescent area after SYTOX green nucleic acid stain. The intracellular reactive oxygen species (ROS) generation was measured by flow cytometry. Total and phosphorylated Syk, SHP-1, and ERK were detected by immunoblot. We found that human monomeric IgG and its subclasses IgG1 and IgG2 per se induced negligible NET formation of dHL-60, but the FcγRIII engagement by these IgG subclasses and Fc portion augment PMA-stimulated dHL-60 NET formation in a dose-dependent manner. Furthermore, we found that increased Syk and ERK phosphorylation, intracellular ROS generation, and pro-inflammatory cytokines, IL-8 and TNF-α, production could be induced after FcγRIII engagement. Blocking FcγRIII engagement by a specific antibody diminished the augmented NET formation. In conclusion, we discovered that cross-talk between FcγRIII engagement-induced Syk-ERK and PMA-induced PKC signaling pathways augment NET formation of dHL-60 via increased ROS generation and pro-inflammatory cytokines, IL-8 and TNF-α, production.

Antibacterial activity of geraniin from sugar maple leaves: an ultrastructural study with the phytopathogen Xanthomonas campestris pv. vitians.

Effect of geraniin extracted from sugar maple (Acer saccharum) leaves on the viability of the phytopathogen Xanthomonas campestris pv. vitians was evaluated with the SYTOX Green nucleic acid stain, penetrating only compromised membranes, and plate counts. In parallel, structural changes of treated bacteria were examined in transmission electron microscopy (TEM). Based on SYTOX Green and plate counts, geraniin at the minimum bactericidal concentration (3.125 mg/ml) increased mortality after 45 min by 37% and 62%, respectively, when compared with controls. According to observations in TEM, geraniin caused morphological alterations of these rod-shaped bacteria, including degradation of their envelopes, as also suggested by the incorporation of SYTOX. These alterations were often accompanied by cytoplasm leakage and the formation of more pronounced whitish areas in the cytoplasm similar to vacuolization. Moreover, multi-membranous and/or –wall systems were at times formed in the treated bacteria. The presence of some extracellular electron-dense material was frequently noted around the treated bacteria. The matrix surrounding control bacteria tended to disappear after geraniin treatment. This study highlights for the first time the effect of geraniin on bacterial ultrastructure, thus contributing to a better understanding of the mechanism by which this molecule exerts antibacterial activity.

The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure to PEF indicate a strong coupling between these parts of the cell.

Superbright organosilica nanodots as a universal sensor for fast discrimination and accurate quantification of live/dead cells

Rapid and quantitative discrimination between live and dead cells is highly desirable yet remains a challenging task. Here, we report the use of the ultrabright green-emitting organosilica nanodots (OSiNDs, photoluminescence quantum yield: ˜100%) for fast (1 min in vitro and 5 min in vivo), wash-free, and universal labeling of the dead bacterial, fungal, and mammalian cells, which meets the demand of the green fluorescent probes that can specifically image the dead cells. The photostability of OSiNDs surpasses that of the commercial dyes, such as propidium iodide (PI) and SYTOX Green nucleic acid stain (SYTOX), making OSiNDs more suitable for long-term and stable monitoring of the cell viability. With the assistance of a commercial red fluorescent dye SYTO 60, we develop an in situ method for rapid and accurate quantification of live/dead cells. Motivated by the unique optical advantages and high sensitivity of OSiNDs to the cell viability, the OSiNDs are employed to observe the in vivo cellular changes induced by doxorubicin using zebrafish as a model, demonstrating their great potential for evaluating the toxicity/efficacy of the therapeutic agents in real time. Owing to the excellent biocompatibility and low cost, the OSiNDs can be a universal probe for efficient and accurate differentiation and quantification of the live and dead cells, which may find wide applications in the biomedical field.

N-Acetylcysteine As a Therapeutic for JAK2V617F Myeloproliferative Neoplasms

Chronic inflammation is common in MPN and drives disease progression and worsens symptom burden. It has been reported that JAK2V617F hematopoietic stem and progenitor cells (HSPCs) exhibit elevated basal oxidative stress compared to wild-type cells. However, JAK2V617F HSCs have a differential response than normal HSPCs upon stimulation with pro-inflammatory cytokines or lipopolysaccharide (LPS) invivo and in vitro. Specifically, JAK2V617F HSPCs do not significantly increase reactive oxygen species, exit quiescence, or increase DNA damage following LPS exposure (Blood 2017 130:4211). Therefore, we hypothesized that oxidative stress is important for differential responses of wild-type and JAK2V617F HSCs to inflammation.To compare cell responses to oxidative stress, we treated bone marrow cells from JAK2V617F knock-in and wild-type mice ex vivo with L-buthionine-S,R-sulfoximine (BSO), which reduces glutathione levels, then measured the impact on myeloid colony formation in methylcellulose. JAK2V617F knock-in bone marrow cells are resistant to oxidative stress-induced reduction in colony formation compared to wild-type bone marrow cells. This data suggests that JAK2V617F myeloid progenitors produce less reactive oxygen species in response to BSO or that these cells are resistant to oxidative stress-induced cell death.Next, we tested the effect of the anti-oxidant n-acetylcysteine (NAC) in a JAK2V617F knock-in mouse model. All hematopoietic cells in this model express JAK2V617F, these mice develop elevated blood counts, splenomegaly, and die suddenly at approximately 2-3 months of age. Surprisingly, addition of NAC (2g/L) in the drinking water extended the lifespan of JAK2V617F-knock in mice (p<0.02, n=6-12). However, aspirin (16mg/L) in drinking water did not extend the lifespan of JAK2V617F-knock in mice. NAC did not alter peripheral blood counts in either JAK2V617F-knock in or wild-type mice, which suggests that NAC's utility in JAK2V617F is not through cytoreduction but is due to its ability to reduce oxidative stress or thrombosis. Additionally, NAC had no effect on thrombin-induced platelet activation, which was assayed by P-selectin expression, phosphatidylserine exposure, and platelet-leukocyte aggregation via flow cytometry.There is a positive correlation between leukocytosis and thrombotic risk in MPN patients. Additionally, MPN patients and mice exhibit elevated neutrophil extracellular trap (NET) formation compared to healthy controls, which contributes to the increased thrombosis in MPN. To test the effect of antioxidants on NET formation in vitro, we treated neutrophils from MPN patients or healthy donors with phorbol myristate acetate concurrently with antioxidants (NAC or ferulic acid) and assayed the presence of extracellular DNA using a SYTOX Green nucleic acid stain. Unstimulated neutrophils from MPN patients exhibited sporadic NET formation while neutrophils from healthy donors did not. Additionally, NAC and ferulic acid reduced DNA release, which is indicative of NET formation.Taken together, these data demonstrate that JAK2V617F progenitors are resistant to oxidative stress-induced cell death. Furthermore, reduction of oxidative stress with n-acetylcysteine in vivo prevented NET-induced thrombosis in JAK2V617F knock-in mice as well as in vitro in normal and MPN neutrophils. These data provide a rationale for investigating the utility of n-acetylcysteine as a therapeutic in myeloproliferative neoplasms. DisclosuresNo relevant conflicts of interest to declare.

Inactivation of invasive marine species in the process of conveying ballast water using [rad]OH based on a strong ionization discharge

In this paper, invasive marine species in medium-salinity ballast water were inactivated using OH generated from a strong ionization discharge. The OH is determined by the concentration of oxygen active species combined with the effects of water jet cavitation. The results indicated that the OH concentration reached 7.62 μM, within 1 s, which is faster and higher than in conventional AOP methods. In a pilot-scale OH ballast water system with a capacity of 10 m3/h, algae were inactivated when CT value was 0.1 mg min/L with a contact time only 6 s. The viable and nonviable cells were determined using SYTOX Green nucleic acid stain and Flow cytometry. As a result, the OH treatment could be completed during the process of conveying the ballast water. In addition, the concentrations of relevant disinfection by-products (DBPs), such as trihalomethanes (THMs), haloacetic acids (HAAs), and bromate, were less than that required by the World Health Organization's drinking water standards, which suggest that the discharged ballast water posed no risks to the oceanic environment. Nevertheless, for conventional ozonation and electrolysis methods, the ballast water should be treated only in the treated tanks during the ship's voyage and form higher level DBPs.

Inhibition of histone deacetylases by trans-cinnamic acid and its antitumor effect against colon cancer xenografts in athymic mice.

Previous studies have shown that trans-cinnamic acid (tCA) has a broad spectrum of biological activities, and exhibits antioxidant, anti-inflammatory and anticancer properties. In addition, tCA and a variety of its analogs have been detected as gut microbe-derived metabolites exerting various biological effects in the colon. The aim of this study was to assess the antitumor activity of tCA in vitro and in vivo, in particular its therapeutic efficacy against colon cancer xenografts in athymic mice. Furthermore, it aimed to examine the effects of tCA on histone deacetylases (HDACs) and to identify the underlying molecular mechanisms. Using an MTT assay, tCA was observed to inhibit the proliferation of several cancer cell lines, and the half maximal inhibitory concentration (IC50) in HT29 colon carcinoma cells was ~1 mM. Western blot analysis demonstrated that tCA upregulated the expression of acetyl-H3 and acetyl-H4 proteins, which was consistent with the effects of the HDAC inhibitor, trichostatin A (TSA). Furthermore, expression of Bcl-2 (a marker of cell proliferation) was reduced, and apoptosis was induced. Apoptosis was shown by the activation of cleavage of poly ADP ribose polymerase and the increased expression of Bax. Apoptosis was also confirmed using APC Annexin V and SYTOX Green Nucleic Acid Stain. In addition, the tCA-induced inhibition of the expression of HDAC markers and activation of apoptosis in tumor tissues were further confirmed by immunohistochemistry. Intragastric administration of tCA at doses of 1.0 and 1.5 mmol/kg body weight suppressed the growth of HT29 human colon carcinoma xenografts in athymic mice at well-tolerated doses. No toxic changes were found in the heart, lung, liver, kidney, colon or bone marrow following histopathological examination. This study indicated that tCA is effective against colon cancer xenograft in nude mice. The antitumor mechanism of tCA was mediated, at least in part, by inhibition of HDACs in cancer cells. As an endogenous microbial metabolite predominantly produced in the colon, tCA is an agent of interest for further evaluation.

Spatio-temporal study of phytoplankton cell viability in a eutrophic reservoir using SYTOX Green nucleic acid stain

Despite the global importance of phytoplankton primary production, the ecological role of cell death as an important loss process in phytoplankton is poorly understood. To assess the significance of cell death in phytoplankton, we studied cell viability of dominant species in the canyon-shaped eutrophic Řimov Reservoir (Czech Republic) at weekly and biweekly intervals from April to October 2011. Surface samples were taken from the lacustrine zone (near the dam, low nutrient level) and transition zone (near the river inflow, high nutrient level) of the reservoir. Moreover, samples from euphotic depth (1% of surface irradiance) were taken from the lacustrine zone. We used the membrane-impermeant nucleic acid dye SYTOX Green to examine seasonal and spatial differences in phytoplankton cell viability. Three species (diatoms Asterionella formosa, Fragilaria crotonensis, and cyanobacterium Aphanizomenon flos-aquae) were studied in detail. There was no difference in Asterionella cell viability among sampling sites. In the lacustrine zone, Fragilaria and Aphanizomenon exhibited lower viability than in the transition zone. In addition, Aphanizomenon viability was significantly lower at the euphotic depth. Nutrient levels were revealed as a factor influencing Fragilaria viability, while light availability was more important for Aphanizomenon. Our results evidenced that the importance of cell death, in particular phytoplankton taxa, varies both spatially and temporally. Moreover, our study indicates that coexisting taxa may differ in their capacity to cope with different environmental stressors.

Cisplatin Fails To Stimulate Production Of Reactive Oxygen Species and Release Of Neutrophil Extracellular Traps By Human Neutrophils In Vitro

Background Cisplatin is a commonly used antineoplastic agent for treatment of a broad range of cancers. Cisplatin-based treatment has been associated with a significant risk of venous thromboembolism. The mechanisms through which cisplatin contributes to a prothrombotic state remain unclear. Neutrophil extracellular traps (NETs) consist of web-like DNA–histone core decorated with granule proteins and are released from activated neutrophils in a process dependent on reactive oxygen species (ROS), in particular hypochlorous acid (HOCl). Recently, NETs have been shown to play an important role in initiation and propagation of venous thrombus in a number of animal models of deep vein thrombosis. The aim of this study was to investigate whether NETs may provide a potential link between cisplatin and venous thromboembolism. Methods and Results To assess the effect of cisplatin on release of NETs by ex vivo human neutrophils isolated by positive immunomagnetic selection we visualised NETs release by confocal fluorescent microscopy and performed fluorimetric quantification of cell-free DNA (CFDNA) using either SYTOX Green nucleic acid stain (10 µM) or an ultrasensitive fluorescent assay Picogreen Quant IT (Invitrogen). In contrast to stimulation with phorbol 12-myristate 13-acetate (PMA) (25 nM),which resulted in 22 ng/104neutrophils of detectable CFDNA, neither of these two assays could detect any significant release of CFDNA by human neutrophils exposed to cisplatin (15 µM) for 2 or 4 hours above baseline similar with vehicle control. Furthermore, confocal fluorescent microscopy imaging of neutrophils stained with non-cell permeable DNA dye SYTOX Red (Invitrogen) demonstrated no difference in NET formation between control and cisplatin treated human neutrophils. Thus we could not demonstrate that NETS are produced in response to cisplatin treatment. In view of consistent reports that NET formation is ROS dependent we decided to investigate whether cisplatin exposure leads to production of ROS by human neutrophils. Few published studies into the effects of cisplatin on the production of ROS by human neutrophils in vitro offer conflicting results. We used flow cytometry and fluorescent probe hydroethidine (HE) for detection of intercellular superoxide anion radical in HL60 granulocytic cells in the presence of cisplatin (up to 50 µM). Differentiation down the granulocytic lineage after stimulation with ATRA was confirmed by light microscopy and by flow cytometry. Capacity of differentiated HL60 cells to generate NET formation after PMA stimulation was confirmed by fluorescence microscopy. Cisplatin failed to augment the spontaneous production of ROS by ATRA differentiated HL60 cells. The number of viable ethidium-high cells in cisplatin treated group did not differ from the vehicle control indicating no detectable production of ROS in response to cisplatin. In contrast, positive control treatment with PMA (25 nM) and menadione (40 µM) resulted in 4- and 20-fold increase in viable ethidium-high population respectively. ROS generation by human neutrophils was measured by a colorimetric assay for chlorination of extracellular taurine to determine if exposure to cisplatin results in the production of HOCl by human neutrophils in vitro. Treatment of resting neutrophils with cisplatin (15 µM) for 30 min or 120 min was not associated with an increase in the spontaneous production of HOCl above the baseline. Furthermore, the PMA (25 nM)-activated generation of HOCl production was not increased by pre-treating neutrophils with cisplatin indicating that there was no potentiation of ROS by pre-treatment with cisplatin. Discussion and Conclusion Our results suggest that cisplatin fails to induce release of NETs or HOCl from human neutrophils in vitro. These negative findings seem to be at odds with the well described pro-oxidative actions of cisplatin. One possible explanation centres on reported findings that the pro-oxidative effects of cisplatin are dependent on the mitochondrial generation of ROS whilst the mitochondria-generated ROS appear not to be instrumental to NET formation. Therefore, we postulate that cisplatin may not be able to induce NET formation by human neutrophils, which are known to contain few mitochondria, due to a sub-threshold ROS signal. Therefore it appears that cisplatin-associated increased risk of venous thrombosis is unlikely to be mediated through NETs. Disclosures: No relevant conflicts of interest to declare.

Antifungal Activity of Resveratrol against Botrytis cinerea Is Improved Using 2-Furyl Derivatives

The antifungal effect of three furyl compounds closely related to resveratrol, (E)-3,4,5-trimethoxy-β-(2-furyl)-styrene (1), (E)-4-methoxy-β-(2-furyl)-styrene (2) and (E)-3,5-dimethoxy-β-(2-furyl)-styrene (3) against Botrytis cinerea was analyzed. The inhibitory effect, at 100 µg ml−1 of compounds 1, 2, 3 and resveratrol on conidia germination, was determined to be about 70%, while at the same concentration pterostilbene (a dimethoxyl derivative of resveratrol) produced complete inhibition. The title compounds were more fungitoxic towards in vitro mycelial growth than resveratrol and pterostilbene. Compound 3 was the most active and a potential explanation of this feature is given using density functional theory (DFT) calculations on the demethoxylation/demethylation process. Compound 3 was further evaluated for its effects on laccase production, oxygen consumption and membrane integrity of B. cinerea. An increase of the laccase activity was observed in the presence of compound 3 and, using Sytox Green nucleic acid stain, it was demonstrated that this compound altered B. cinerea membrane. Finally, compound 3 partially affected conidia respiration.

Aptamer‐mediated complement system killing of MCF‐7 and MEAR cancer cells

Aptamer MUSC1-5TR-1 has high affinity for Mucin 1 (MUC1) which is over expressed in the adenocarcinoma cell line MCF-7. Aptamer TLS-11a recognizes BNL 1ME A.7R.1 (MEAR) hepatocarncinoma cells (Shangguan, D, et al., Anal Chem 80, no. 3: 721–728). Biotinylation of the aptamers' 5′ end allowed conjugation to streptavidin that was in turn conjugated to C1q, the first protein in the classical complement immune response pathway. Killing of MCF-7 and MEAR cells in vitro by complement-mediated lysis resulting from exposure to the biotinylated aptamers-streptavidin-C1q, in the presence of human complement protein serum, was visualized using SYTOX Green nucleic acid stain. SYTOX Green (Invitrogen) brightly labels nucleic acid in cells with compromised membrane integrity. MCF-7 and MEAR cells exposed to the full treatment both showed increased nuclear staining within a few hours, whereas cells not exposed to the aptamer, streptavidin-C1q conjugate, or complement protein serum showed little to no nuclear staining. Future experiments will incorporate FRET detection of membrane potential changes resulting from MAC formation, and will utilize immuno-fluorescence and immuno-gold to localize components of the MAC by laser scanning confocal and electron microscopy. Supported by funding from OTC Biotechnologies, LLC and NSF DBI-0821252 to JK.

Use of SYTOX green dye in the flow cytometric analysis of bacterial phagocytosis.

Fluorescein isothiocyanate (FITC) is used widely to label the targets used in flow cytometric phagocytosis assays. Unfortunately, the fluorescence intensity of phagocytosed FITC-labeled targets is influenced by changes in intracellular pH level, making quantitative measurements with this fluorophore problematic. We describe the use of SYTOX green nucleic acid stain to measure phagocytosis by flow cytometry. Suspensions of isopropyl alcohol-permeabilized Escherichia coli DH5alpha were stained with the SYTOX green dye and then incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytosis was determined by gating the cells. Results are expressed as percentage of phagocyte-associated green fluorescent cells. The validity of the method was shown by the effects of a phagocytosis inhibitor (incubation at 4 degrees C) or enhancer (gamma interferon [IFN- gamma] treatment) being accurately assessed with this assay. The method described was reproducible and provides an advantageous alternative to the use of FITC to label bacteria for the flow cytometric measurement of target uptake by phagocytic cells.

Differentiation of Viable and Dead Escherichia coli O157:H7 Cells on and in Apple Structures and Tissues following Chlorine Treatment

Confocal scanning laser microscopy (CSLM) was used to differentiate viable and nonviable cells of Escherichia coli O157:H7 on and in raw apple tissues following treatment with water and 200 or 2,000 ppm active chlorine solution. Whole unwaxed Red Delicious cultivar apples at 25°C were inoculated by dipping in a suspension of E. coli O157:H7 (8.48 log10 CFU/ml) at 4°C, followed by treatment in water or chlorine solution at 21°C for 2 min. The dead cells on and in apples were distinguished from live cells by treating tissue samples with SYTOX green nucleic acid stain. Viable and dead cells were then labeled with an antibody conjugated with a fluorescent dye (Alexa Fluor 594). The percentage of viable cells on the apple surface, as well as at various depths in surface and internal structures, was determined. The mean percentages of viable cells located at the sites after treatment with water or chlorinated water were in the following order, which also reflects the order of protection against inactivation: floral tube wall (20.5%) > lenticels (15.0%) > damaged cuticle surrounding puncture wounds (13.0%) > intact cuticle (8.1%). The location of viable cells within tissues was dependent on the structure. Except for lenticels, the percentage of viable cells increased as depth into the CSLM stacks increased, indicating that cells attached to subsurface structures were better protected against inactivation with chlorine than were cells located on exposed surfaces. Further research is warranted to investigate the efficacy of other chemical sanitizers, as well as that of surfactants and solvents in combination with sanitizers, in removing or killing E. coli O157:H7 lodged in protective structures on the surface and within tissues of apples.

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-α, 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-α using monoclonal anti-human TNF-α antibody and determined that ∼20 ng/mL antibody was sufficient to neutralize 50% of the biological activity of 250 pg/mL rhTNF-α 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.

Effectiveness of SYTOX Green stain for bacterial viability assessment.

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of Escherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.