Discrimination of DNA and RNA in cells by a vital fluorescent probe: lifetime imaging of SYTO13 in healthy and apoptotic cells

BACKGROUND: Of the few vital DNA and RNA probes, the SYTO dyes are the most specific for nucleic acids. However, they show no spectral contrast upon DNA or RNA binding. We show that fluorescence lifetime imaging using two-photon excitation of SYTO13 allows differential and simultaneous imaging of DNA and RNA in living cells, as well as sequential and repetitive assessment of staining patterns. METHODS: Two-photon imaging of SYTO13 is combined with lifetime contrast, using time-gated detection. We focus on distinguishing DNA and RNA in healthy and apoptotic Chinese hamster ovary cells. RESULTS: In healthy cells, SYTO13 has a fluorescence lifetime of 3.4 +/- 0.2 ns when associated with nuclear DNA. Bound to RNA, its lifetime is 4.1 +/- 0.1 ns. After induction of apoptosis, clusters of SYTO13 with fluorescence lifetime of 3.4 +/- 0.2 ns become apparent in the cytoplasm. They are identified as mitochondrial DNA on the basis of colocalization experiments with the DNA-specific dye, DRAQ5, and the mitochondrial-specific dye, CMXRos. Upon progression of apoptosis, the lifetime of SYTO13 attached to DNA shortens significantly, which is indicative of changes in the molecular environment of the dye. CONCLUSIONS: We have characterized SYTO13 as a vital lifetime probe, allowing repetitive and differential imaging of DNA and RNA.     

Towards an understanding of apoptosis detection by SYTO dyes.

BACKGROUND: SYTO probes are gaining momentum as reliable and easy to use markers of apoptotic cell death, but the phenomenon underlying reduced SYTO fluorescence in apoptotic cells as compared with normal cells is still not fully elucidated. Herein, we attempt to provide further insights into mechanisms of reduced SYTO16 fluorescence during apoptosis. METHODS: Human follicular lymphoma cell lines were subjected to diverse apoptotic and oncotic stimuli with subsequent multiparametric flow cytometric and fluorescence imaging analysis. SYTO green (SYTO11-16), TMRM, PI, 7AAD, and Hoechst 33342 probes were applied for multivariate analysis of temporal sequence of apoptotic events. Sorting of cells differing in the level of SYTO16 fluorescence and subsequent characterization of obtained subpopulations were also performed. RESULTS: Loss of SYTO16 fluorescence (SYTOlow/PI+ events) has been observed in cells exposed to oncotic stimuli, whereas SYTOhigh/PI+ events did not prevail at any treatment scenario. We tracked similarities and discrepancies between SYTO16 and TMRM probes. Often, SYTO16 and TMRM exhibited the same staining profiles, as loss of their fluorescence was detected in a single cell population. However, both mitochondrial uncoupler FCCP and a small-molecule Bcl-2 inhibitor, HA14-1, appeared to induce distinct staining profiles of SYTO16 and TMRM, with the decrease in TMRM fluorescence preceding the loss of SYTO16 fluorescence. Importantly, in both cases (FCCP and HA14-1) the decrease of SYTO16 fluorescence was blocked by pharmacological inhibition of caspases (with z-VAD-fmk). CONCLUSIONS: The data demonstrate that loss of SYTO16 is caspase-dependent, as is not a mere indicator of Deltapsim dissipation. Commonly observed similarities between SYTO and TMRM may stem from the fast kinetics of apoptotic events once cell death is initiated.     

Flow cytometric analysis of apoptotic subpopulations with a combination of annexin V-FITC, propidium iodide, and SYTO 17

BACKGROUND: We have previously characterized apoptotic cell death induced in a follicular lymphoma cell line, HF-1, after triggering via the B-cell receptor (BCR) or treatment with Ca(2+) Ionophore A23187. We analyzed the kinetics of apoptosis induced by these two treatments, as two alternative models of classical apoptosis, by flow cytometry using a novel combination of cytofluorometric stains. METHODS: Cells were stained with a combination of Annexin V-FITC, propidium iodide (PI), and SYTO 17 and analyzed by a two-laser flow cytometry system using 488-nm argon and 633-nm HeNe air-cooled lasers. RESULTS: In both apoptotic models, the first apoptotic cells were detected by SYTO 17 staining. The alteration in SYTO 17 staining intensity was followed by an increased uptake of PI. Finally, the apoptotic cells were labeled with Annexin V in BCR-induced apoptosis. On the contrary, on treatment with Ca(2+) Ionophore A23187, cells became positive for Annexin V earlier than for PI. CONCLUSIONS: The novel cytofluorometric dye, SYTO 17, discriminates apoptotic alterations before Annexin V and PI. PI also discriminates apoptotic alterations before the loss of plasma membrane asymmetry by BCR but not by Ca(2+) Ionophore A23187-induced apoptosis. Finally, the combination of these three cytofluorometric dyes allows effective detection of apoptotic subpopulations and ordering of apoptotic events by flow cytometry.     

Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing's sarcoma cells with a vital fluorescent probe

A fluorescence lifetime imaging microscope (FLIM) was applied to study early-stage apoptotic cells stained with a SYTO13 dye. The fluorescence lifetime of SYTO13 in healthy cells was 3.8+/-0.3ns but was reduced to 2.4+/-0.4 and 1.9+/-0.2ns after a 3-h period of incubation with SYTO13 when doxorubicin, a known inducer of apoptosis, was added to human Ewing's family tumor cells at final concentrations of 250 and 500nM, respectively, in a dose-dependent experiment. On the other hand, in a time-dependent experiment, the fluorescence lifetime decreased to 2.5+/-0.5 and 1.7+/-0.4ns at a doxorubicin concentration of 750nM after 2 and 4h, respectively. A possible explanation for these results is self-quenching induced by a change in interprobe distance that arises from the condensation of DNA during apoptosis. In this study, the FLIM system was employed to investigate early-stage apoptosis that involves only small morphological changes, suggesting the potential advantage of this method for evaluating small biological effects in living cells.     

Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain-no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry.Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors.     

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4'',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.     

Transient and sustained oxidative stress differentially activate the JNK1/2 pathway and apoptotic phenotype in H9c2 cells

The aim of this study was to investigate the activation of JNK1/2 signalling pathway and the respective cellular phenotype of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation of JNK1/2 pathway by exogenous H₂O₂, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation of c-Jun was observed, coincidently with the activation of nuclear JNK1/2; under sustained stress, these phosphorylation levels remained elevated above basal for up to 6 h, whereas under transient stress they declined to basal ones within 4 h of withdrawal. Furthermore, the JNK1/2 selective inhibitor SP600125 abolished the c-jun phosphorylation induced by oxidative stress. Our results using cell viability assays and light microscopy revealed that sustained H₂O₂ stimulation significantly and time-dependently decreased H9c2 viability, in contrast to transient stimulation; SP600125 (10 μM) abolished cell death induced by sustained as well as cell survival induced by transient oxidative stress. Hoechst staining showed an increase in DNA condensation during sustained, but not during transient stimulation. Moreover, from the antioxidants tested, catalase and superoxide dismutase prevented oxidative stress-induced cell death. Flow cytometry studies reconfirmed that sustained oxidative stress induced apoptosis, whereas transient resulted in the recovery of cardiac myoblasts within 24 h. We conclude that in H9c2 myoblasts, sustained activation of JNK1/2 signalling pathway during oxidative stimulation is followed by an apoptotic phenotype, while transient JNK1/2 activation correlates well with cell survival, suggesting a dual role of this signalling pathway in cell fate determination.     

Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.     

Cytosine Arabinoside Induces Apoptosis in Cerebellar Neurons in Culture

Abstract: Cytosine arabinoside (AraC) is a pyrimidine antimetabolite that prevents cell proliferation by inhibiting DNA synthesis. We report that AraC kills cultured cerebellar neurons in a concentration-dependent fashion with an EC₅₀ of ∼60 µ M when added shortly after seeding. This cell death has apoptotic features because we observed (1) morphology of apoptotic nuclei as judged by DNA staining with Hoechst 33258, (2) DNA fragmentation with typical ladder pattern on agarose gel, (3) positive nuclear labeling with a specific in situ DNA fragmentation staining, (4) prevention by deoxycytidine (IC₅₀ = 1 µ M ), protein, and RNA synthesis inhibitors, and (5) release of DNA fragments in the incubating medium. We have also observed that several proteins were overexpressed in AraC-treated neurons by two-dimensional polyacrylamide gel electrophoresis. We conclude that AraC induces a signal that triggers a cascade of new mRNA and protein synthesis, leading to apoptotic cell death in cultured cerebellar granule cells.     

Phototoxicity of the fluorescent membrane dyes PKH2 and PKH26 on the human hematopoietic KG1a progenitor cell line.

BACKGROUND: The phototoxic effects of the well-known fluorescent membrane dyes PKH2 and PKH26 have been unknown, although their use in cell tracking experiments has increased dramatically. To eliminate the phototoxicity-induced alteration in cell function and morphology, it is essential to examine the suspicious phototoxicity of these dyes. METHODS: Chemical and phototoxic effects of PKH dyes on the human hematopoietic KG1a cell line were examined. To minimize phototoxicity in long-term cell tracking experiments lasting up to 18 h with a fluorescence microscope system, time-lapse monitoring with different time intervals and exposure times was introduced. RESULTS: There were no significant effects of the two PKH dyes on cell viability and growth when using dye concentrations up to 5 microM. However, when stained cells were exposed to excitation light, cell viability decreased dramatically, showing the phototoxicity of the PKH dyes. More than 60% of cells stained with 5 microM PKH26 died after 5 min of continuous light exposure. The phototoxic effect was more extensive in cells stained with higher concentrations of the dyes. CONCLUSIONS: We present guidelines for the optimal use of these dyes by using a defined hardware configuration.     

Apoptotic Cell Death and Caspase-3 Activation Induced by N-Methyl-d-Aspartate Receptor Antagonists and Their Prevention by Insulin-Like Growth Factor I

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.     

Staurosporine-induced neuronal death: multiple mechanisms and methodological implications

To examine whether multiple pathways of cell death exist in sympathetic neurons, we studied the cell death pathway induced by staurosporine (STS) in sympathetic neurons and compared it with the well-characterized NGF deprivation-induced death pathway. Increasing concentrations of STS were found to induce sympathetic neuronal death with different biochemical and morphological characteristics. One hundred nM STS induced metabolic changes, loss of cytochrome c, and caspase-dependent morphological degeneration which closely resembled the apoptotic death induced by NGF deprivation. In contrast, sympathetic neurons treated with 1 microM STS showed no loss of cytochrome c but exhibited extensive, caspase-independent, chromatin changes that were not TUNEL positive. One microM STS-treated sympathetic neurons had greatly reduced metabolic activities and became committed to die rapidly, yet maintained soma structure and appeared viable by other criteria even up to 48 h after STS treatment, illustrating the need to assess cell death by multiple criteria. Lastly, in contrast to the cell death-inducing activities of 100 nM STS or 1 microM STS, very low concentrations of STS (1 nM STS) inhibited sympathetic neuronal death by acting either at or prior to c-jun phosphorylation in the NGF deprivation-induced PCD pathway.     

Osteoprotegerin protects endothelial cells against apoptotic cell death induced by Porphyromonas gingivalis cysteine proteinases

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis during the progression of periodontitis. Recent reports suggest that osteoprotegerin may also prevent arterial calcification and contribute to endothelial cell survival. To determine whether the vascular functions of osteoprotegerin are involved in periodontitis, we examined whether osteoprotegerin contributed to the survival of endothelial cells damaged by Porphyromonas gingivalis cysteine proteinases (gingipains). Gingipain proteinases cleave a broad range of host proteins, and are important virulence factors of P. gingivalis, a major causative bacterium of adult periodontitis. Human microvascular endothelial cells (HMVEC) were exposed to activated gingipain extracts from P. gingivalis 381, with and without pretreatment with osteoprotegerin. Cell viability was quantified by the tetrazolium (WST-8) reduction assay, and apoptosis was examined using Hoechst 33342 nuclear staining. After 16 h of treatment with activated gingipain extracts, HMVEC showed near-complete detachment from the tissue culture dish, and apoptosis was evident by 24 h. Pretreatment of HMVEC with osteoprotegerin reduced the extent of both cellular detachment and apoptotic cell death. Our results indicated that osteoprotegerin pretreatment protected HMVEC against detachment and apoptotic cell death induced by gingipain-active bacterial cell extracts. These results also suggest that osteoprotegerin may function as a survival factor for endothelial cells during periodontitis.     

Laser scanning cytometry in the characterization of the proapoptotic effects of transiently transfected genes in cerebellar granule neurons.

BACKGROUND: Low transient transfection efficiency limits the ability to characterize putative proapoptotic gene function in neurons. Laser scanning cytometry (LSC), with its high capacity, medium throughput means of collecting fluorescent emissions from cultured cells, offers an effective technology for scoring cell death in neuronal transfectants. METHODS: Cerebellar granule neurons (CGNs) were transfected with EGFP-fusion constructs of Caspase-3 and Caspase-9 using a DNA-calcium phosphate coprecipitation method. CGNs were fixed, permeablized, and stained with propidium iodide (PI) nuclear dye. An LSC method, based on a combination of Long Red Max Pixel, Long Red Integral, and Green Integral fluorescence parameters was validated for the scoring of apoptotic cell death in CGNs. RESULTS: In Caspase-3 and Caspase-9 transfected CGNs, cell death was scored both in transfectants and nontransfected culture-mates. The cell death phenotype was found to be independent of transfection efficiency. LSC scoring of Caspase-9 transfectants was compared with visual scoring following Hoechst 33342 staining, yielding results that were similar qualitatively, but not quantitatively, likely owing to the greater sensitivity to green fluorescence of laser scanning compared to human vision. CONCLUSION: LSC scoring of transiently transfected CGNs offers a rapid and reliable means of characterizing proapoptotic gene effects.     

Paclitaxel induces apoptosis in Saos-2 cells with CD95L upregulation and Bcl-2 phosphorylation.

We examined the effect of paclitaxel on human osteoblastic cells Saos-2 to determine if paclitaxel can affect proliferation and apoptosis. We used a p53-negative cell line in order to mimic the loss of function frequently observed at the clinical level. Paclitaxel induced cell death in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were observed by Hoechst 33258 stain, DNA ladder formation, electron microscopy, and flow cytometry at concentrations as low as 100 nM, a concentration which can be achieved by infusion in human plasma. At 100 nM, paclitaxel induced a G2 arrest at 8 h of treatment. The cells then continued to accumulate in G2 until 72 h when the percentage of apoptotic events reached 54%. At the molecular level, Bcl-2 protein was phosphorylated at 16 h and PARP protein was cleaved, indicating the activation of caspase-3-like proteases. Caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK rescued Saos-2 cells from paclitaxel-induced apoptosis. CD95 expression was constantly high, while CD95L showed a threefold increase in expression. This suggests that, following the G2 arrest, apoptosis is induced through the CD95/CD95L system.     

Defining viability in mammalian cell cultures

A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure membrane integrity (a measure of necrotic cell death) and assays that measure apoptotic cells, and show that discrepancies in the measurement of culture viability have a significant impact on the calculation of cell culture parameters and lead to skewed experimental data.     

Neuroprotection of Insulin against Oxidative Stress-induced Apoptosis in Cultured Retinal Neurons： Involvement of Phosphoinositide 3-kinase/Akt Signal Pathway

In order to investigate the neuroprotection of insulin in retinal neurons,we used retinal neuronalculture as a model system to study the protective effects of insulin against H_2O_2-induced cytotoxicity andapoptotic death.Primary retinal neuronal cultures were grown from retinas of 0-2-day old Sprague-Dawleyrats.Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay,and byDNA laddering analysis.Phosphoinositide 3-kinase (PI3K) activity was measured using phosphoinositide4,5-bisphophate and [γ-~(32)P]ATP as substrate.Western blot analysis with anti-phospho-Akt (pS473) antibodywas performed to examine the level of phosphorylated Akt.We observed that treatment with 100μM H_2O_2for 24 h significantly decreased cell viability and induced apoptotic death of retinal neurons,and that pretreatmentwith 10 nM insulin significantly inhibited or attenuated H_2O_2-induced cytotoxicity and apoptosis.Pretreatmentwith LY294002,a specific PI3K inhibitor,abolished the cytoprotective effect of insulin.Insulin also stronglyactivated both PI3K and the downstream effector Akt.These results suggest that insulin protects retinalneurons from oxidative stress-induced apoptosis and that the PI3K/Akt signal pathway is involved in insulin-mediated retinal neuroprotection.