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Keywords:

  • apoptosis;
  • reduced glutathione;
  • GSH;
  • VitaBright-43;
  • VitaBright-48;
  • monochlorobimane;
  • thiol probe

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

An early step in apoptosis is extrusion of reduced glutathione (GSH). Current assays for measuring apoptosis involve a number of incubation and washing steps, making them time consuming and laborious. Using two novel thiol reactive agents (VitaBright-43 and VitaBright-48) and a GSH specific probe; monochlorobimane, we investigated whether changes in the level of free thiols can be used as an apoptotic marker. Upon addition to cells the probes permeate the cell membrane and react with intracellular thiols, causing cellular fluorescence. Cytometric quantification of the cell fluorescence (without washing) can then be used to determine the population's cellular thiol level at the single cell level. Apoptotic traits such as phosphatidylserine externalisation, caspase activity and mitochondrial potential were investigated at different time points after induction of apoptosis and correlated to changes detected using the thiol probes. We found that though all three thiol probes could be used to detect changes in the level of free thiols correlating well with apoptotic markers, other properties such as detection of early versus late apoptosis and staining kinetics differed among the three probes. However, we suggest adding evaluation of the level of free thiols to the list of phenotypes which may be measured in order to detect apoptosis, as this provides a reliable and easy way of assaying apoptosis. © 2013 International Society for Advancement of Cytometry


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

Apoptosis is a highly regulated process characterised by a number of distinct biochemical and morphological changes such as externalization of phosphatidylserine, activation of caspases, chromatin condensation, and DNA fragmentation. These changes can be used to identify and quantify apoptotic cells. As cell death occurs through different mechanisms, which may be present in various combinations, the Nomenclature Committee on Cell Death (NCCD) encourages researchers to quantify all types of cell death (including apoptosis) with more than one assay, thereby reducing the probability of artifacts [1]. Expanding the list of apoptotic phenotypes does not only provide new insight into the details of the apoptotic machinery, but will also enable new combinations of complementing assays.

It has long been known that treatments which either directly or indirectly induce oxidative stress also possess the ability to induce apoptosis [2]. For example, exposure to low doses of H2O2 has been reported to induce apoptosis in a variety of cell types [3, 4]. More recently the role of redox status alterations in apoptosis induction has been recognized [5, 6]. Cellular redox potential is largely determined by reduced glutathione (GSH) which accounts for more than 90% of cellular nonprotein thiols [7], whereas the oxidized form, glutathione disulfide (GSSG), normally represents <2% of the total glutathione pool [8]. GSH provides a large proportion of the reducing power available in the cell, and its oxidation status largely determines the thiol-disulfide status of the cell by interchange reactions. The concentration of GSH has been found to decrease upon induction of apoptosis, also when using non-oxidative apoptogenic agents, due to extrusion of GSH [9-12]. The decrease in intracellular GSH concentration has been demonstrated in response to very different apoptotic stimuli, including death receptor activation, induction of stress and drug induced cell death [6, 13-16]. Depletion of GSH by the use of an inhibitor of GSH synthesis (l-buthionine sulphoximine, BSO), has also been reported to induce apoptosis [17, 18]. As GSH is a very strong antioxidant, it is not surprising that depletion of GSH causes a raise in the level of ROS. However, the increase in ROS happens after induction of apoptotic signaling mediators, therefore the induction of apoptosis seems to occur as a direct consequence of GSH depletion and not indirectly due to ROS-induced cellular damages [19]. While changes in the oxidative environment may lead to apoptosis, it is not possible to abolish apoptosis by the presence of e.g., pyrovate and the superoxide dismutase mimic MnTBAP; these antioxidants may prevent ROS formation, but not GSH extrusion and apoptosis [9, 19]. It thus is clear that GSH and GSH extrusion play a role in apoptosis beyond that of an antioxidant.

Interchange reactions links the cellular concentration of GSH directly to the level of other reduced thiols. Moreover; the level of GSH, and hence the level of thiols, decreases in response to induction of apoptosis, thus measuring the level of free thiols may be used to quantify apoptosis. We recently explored this using the novel probe, VitaBright-48, which becomes fluorescent after reaction with thiols [20]. Cells induced to undergo apoptosis using either Tumor Necrosis Factor α (TNF α) or camptothecin (CPT) were stained using VitaBright-48 and the fluorescence intensity measured. We found that though apoptotic cells exhibited reduced fluorescence intensity, the decrease in fluorescence occurred after externalisation of phosphotidylserine and activation of caspase 3. As extrusion of GSH is reported to happen at the onset of apoptosis, we had expected also to see reduced fluorescence intensity of VitaBright-48 stained cells shortly after apoptosis induction. In our further investigations reported here, we have explored the properties of the thiol probe monochlorobimane, which reacts with GSH by a reaction catalysed by glutathione S-transferase thereby forming fluorescent adducts. However, we soon realized that this dye had some major limitations as outlined in this article and further investigations led us to the development of another thiol reactive probe, VitaBright-43. We here present data showing that this novel probe provides an easy method for early detection of apoptosis, and compare the properties of VitaBright-43, VitaBright-48 and monochlorobimane for apoptosis detection.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

Cell Lines

Jurkat cells (human leukemia cell line, subclone A3, ATCC CRL-2570), WEHI-S cells (murine fibrosarcoma cell line) [21], and HeLa cells (epithelial cells, cervix, ATCC CCL-2) were cultured in DMEM GlutaMAX-1 medium (GIBCO, Life Technologies, Carlsbad, CA) supplemented with 6% heat-inactivated fetal calf serum (GIBCO). The cells were incubated at 37°C, 5% CO2 atmosphere. Apoptosis was induced by treating Jurkat cells with camptothecin (CPT, Calbiochem, La Jolla, CA), WEHI-S cells with human TNF α (Abcam, UK) and HeLa cells with hydrogen peroxide (SSI Diagnostica, Hilleroed, Denmark) for the time and at the concentrations specified. Depletion of GSH was obtained by treatment of HeLa cells with 0.5 mM BSO (l-buthionine sulphoximine, Sigma-Aldrich) for 16 h. PBS (vehicle) served as control.

Image Cytometry System

Cellular fluorescence was quantified using a NucleoCounter® NC-3000™ image cytometer (ChemoMetec, Alleroed, Denmark). In brief, this instrument combines large field-of-view and depth of focus optics with a high speed imaging system, thereby facilitating high content cell analysis. Depth of focus is more than 100 µm and the field of view is 3.28 mm × 2.45 mm, with a pixel resolution of 2.35 µm/pixel. The magnification is approximately 2X. The NucleoCounter NC-3000 used in this study has seven LED light sources (peak wave lengths at 365, 405, 455, 475, 500, 525, and 625 nm) and a dark field light source used for cell image segmentation. Further details of the NucleoCounter NC-3000 design and specifications can be found at www.chemometec.com. The accompanying NucleoView® NC-3000™ software facilitates automated image acquisition, image analysis, and data visualization and enables quantification and gating of subpopulations. The FlexiCyte™ module enable user-adaptable protocols, which is used to set excitation times for selected light sources, and setting the minimum number of cells (events) to be analyzed. In the assays used here the minimum number of analysed cells was set to 5,000.

Externalization of Phosphatidylserine

Changes in the phosphatidylserine symmetry were determined using annexin V conjugated to CF-647 (Biotium, Hayward, CA) and nonviable cells were stained with SYTOX Green (Invitrogen, Life Technologies, Carlsbad, CA). Briefly, control or apoptosis induced cells were incubated with CF-647 conjugated annexin V according to manufacturer's recommendations. After washing, cells were stained with 0.5 µM SYTOX Green and a thiol probe; either 10 µM VitaBright-43 (ChemoMetec) or 10 µM VitaBright-48 (ChemoMetec) or 10 µM monochlorobimane (SigmaAldrich, St. Louis, MO). Cells stained with monochlorobimane were incubated for exactly 5 min at room temperature before image cytometry analysis, while cells stained with VitaBright-43 and VitaBright-48 were analyzed immediately. Image cytometry was carried out using a NucleoCounter NC-3000 and accompanying software. Segmentation was based on events detected using the dark field light source, and aggregates consisting of more than five cells were excluded. Debris was gated out based on area (pixel coverage) and intensity. CF-647 fluorescence was detected using peak excitation at 625 nm and emission at 740/60 nm, SYTOX Green using peak excitation at 475 nm and emission at 560/35 nm and VitaBright-48 using peak excitation at 405 nm and emission at 475/25 nm, while VitaBright-43 and monochlorobimane were detected at peak excitation at 365 nm and emission at 470/55. The experiment was performed in triplicate.

Fluorochrome-Labeled Inhibitors of Caspases Assay

The Fluorochrome-Labeled Inhibitors of Caspases Assay (FLICA) was used to measure caspase activity in CPT treated Jurkat cells. Briefly, control or CPT-treated cells in cell culture media were incubated with reconstituted FLICA reagent (SR-DEVD-FMK, Immunochemistry, Bloomington, MN) for 60 min at 37°C according to manufacturer's instructions. After washing, cells were stained with a thiol probe; either 10 µM VitaBright-43 or 10 µM VitaBright-48 or 10 µM monochlorobimane as described above. Stained cells were analyzed using the NucleoCounter NC-3000 system. Based on cell data from the dark field channel, image segmentation was performed as described in the above section. The red fluorescent SR-FLICA probe was detected using peak excitation at 525 nm and emission at 675/80 nm and VitaBright-48, VitaBright-43, and monochlorobimane as described above. The experiment was performed three times.

NucView 488 Caspase 3 Assay

Untreated and TNF α treated WEHI-S cells were incubated with 2.5 µM caspase 3/7 specific DEVD-NucView 488 substrate (Biotium, Hayward, CA) for 15 min at room temperature according to manufacturer's instructions. Next, cells were stained with RedDot2 (nonviable stain from Biotium, Hayward, CA, concentration not specified) diluted 1,000 times and a thiol probe; either 10 µM VitaBright-43 or 10 µM VitaBright-48 or 10 µM monochlorobimane as described above. Stained cells were analyzed using the NucleoCounter NC-3000 system. Based on cell data from the dark field channel, image segmentation was performed as described in the above section. NucView 488 was detected using peak excitation at 475 nm and emission at 560/35 nm, RedDot2 was detected using peak excitation at 625 nm and emission at 740/60 and VitaBright-48, VitaBright-43, and monochlorobimane were measured as described above. The experiment was performed in triplicate.

Mitochondrial Potential Measurements

Collapse of the mitochondrial potential was detected using the stain JC-1 (5, 5, 6, 6-tetrachloro-1, 1, 3, 3-tetraethylbenzimidazol-carbocyanine iodide). In brief, control or CPT treated cells were added 2,5 µg/mL JC-1 (Solution 7, ChemoMetec) and incubated at 37°C for 10 min. Then cells were washed twice in PBS and stained with a thiol probe (VitaBright-43, VitaBright-48, or monochlorobimane) as described above. Cells were analyzed using the Mitochondrial Potential Assay on a NucleoCounter NC-3000 (ChemoMetec) combined with detection of VitaBright-48, VitaBright-43 and monochlorobimane using the excitation and emission settings described above.

Staining Kinetics

Kinetics of staining with thiol probes were investigated by adding a mixture of acridine orange (0.5 µM), propidium iodide (50 µM) and a thiol probe; either 10 µM VitaBright-43, 10 µM VitaBright-48 or 10 µM monochlorobimane to cells and then measure the fluorescence intensity by image cytometry after 1, 3, 5, and 10 min incubation using the Vitality Assay on a NucleoCounter NC-3000 (ChemoMetec) with excitation and emission settings for VitaBright-43, VitaBright-48, or monochlorobimane as described above. AO staining was used for cell segmentation. Nonviable cells (PI positive cells) were excluded from the analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

Correlation of Apoptotic Markers and Thiol Probe Staining

Depletion of reduced glutathione is a key event in the apoptotic cascade; thus measuring changes in the level of reduced thiols, reflecting the level of GSH, could be used to assay apoptosis. To explore this we have measured changes in the thiol/GSH level of CPT treated Jurkat cells utilizing three different probes, which upon reaction with GSH/thiols become fluorescent and correlated these changes with three well-known apoptotic markers; phosphatidylserine externalization, caspase 3/7 activity, and loss of mitochondrial transmembrane potential.

Figure 1 follows changes in phosphatidylserine externalisation (annexin V-binding) versus fluorescence intensities of the three thiol probes; VitaBright-43, VitaBright-48, and monochlorobimane in Jurkat cells treated with CPT. At the 3.5-h time point a subpopulation of Jurkat cells induced to undergo apoptosis showed increased phosphatidylserine externalisation combined with a decrease in staining with VitaBright-43 and monochlorobimane. However, the fluorescence intensity of cells stained with VitaBright-48 first decreased after 5 h. No distinct subpopulation could be identified for the VitaBright-48 stained cells; instead the decrease in fluorescence intensity merely broadens the scatterplot to the left. Figure 1 shows, in accordance with the findings previously reported [20], that cells with decreased VitaBright-48 fluorescence are annexin V positive. Nonviable cells also showed low thiol probing staining, but these cells were gated out from the scatter plots in Figure 1.

image

Figure 1. Scatter plots showing changes in phosphatidylserine externalization and staining with VitaBright-43, Vitabright-48 or monochlorobimane in response to treatment with 5 µM or 25 µM CPT versus control after 0.5–5 h. Nonviable cells were gated out based on SYTOX green uptake.

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Figure 2 shows staining with the three thiol probes versus caspase 3/7 activity measured using a sulforhodamine-FLICA probe. An increase in caspase 3/7 activity can be detected at the 3.5 h time point, concurrently with phosphatidyl serine externalisation (Fig. 1) and loss of staining with VitaBright-43 and monochlorobimane. VitaBright-48 fluorescence intensity first decreases later (at the 5 hour timepoint).

image

Figure 2. Scatter plots showing changes in caspase activity (SR-DEVD-FLICA) and staining with VitaBright-43, Vitabright-48 or monochlorobimane in response to treatment with 5 µM or 25 µM CPT versus control after 0.5–5 h.

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The correlation between thiol probe staining and mitochondrial depolarisation (loss of red fluorescence of JC-1 stained cells) are shown in Figure 3. Cells with decreased red fluorescence (apoptotic cells) also exhibit weak thiol probe staining, whether stained with VitaBright-43, VitaBright-48 or monochlorobimane. However, the decrease in staining with VitaBright-48 seems to happen after loss of the mitochondrial potential.

image

Figure 3. Scatter plots showing changes in mitochondrial potential (red fluorescence of JC-1) versus staining with VitaBright-43, Vitabright-48 or monochlorobimane in response to treatment with 5 µM or 25 µM CPT versus control after 0.5–5 h.

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In order to investigate whether the findings described above also comply for other apoptosis models, we furthermore examined the thiol probe staining of WEHI-S induced to undergo apoptosis with TNF α and HeLa cells treated with hydrogen peroxide.

To investigate apoptosis progression in WEHI-S treated with TNF-α, we used the NucView 488 caspase 3 stain. This stain was combined with the RedDot2 dye to exclude non-viable cells and either VitaBright-43, VitaBright-48 or monochlorobimane to quantify changes in the thiol level. Scatterplots for NucView488 fluorescence intensity of viable cells (y-axis) versus their thiol probe fluorescence intensity (x-axis) were divided into four quadrants as outlined in Figure 4a. Examples of the scatter plots can be found in supplementary material Supporting Information Figure S1a. In order to compare performance of the three thiol probes, we performed cytometric analysis in triplicate and graphed the percentage of cells found in each quadrant as a function of time of drug treatment (Figs. 4b–4e). From these graphs it is evident that the level of caspase 3 positive cells increases concurrently with a decrease in Vitabright-43 and monochlorobimane staining, while the decrease in Vitabright-48 staining is delayed and is less profound. Thus, only an insignificant fraction of the caspase 3 positive cells has a high level of Vitabright-43 and monochlorobimane staining, while a high proportion of the caspase 3 positive cells have high levels of Vitabright-48 (Panel c, i.e. upper-right quadrant). The cumulative data from these cytometric analyses confirms that Vitabright-43 and the monochlorobimane thiol probes correlates well with caspase 3 activity and are superior to Vitabright-48 for detecting early apotosis.

image

Figure 4. Dynamics of caspase 3 activity (NucView 488) and thiol probe fluorescence (either VitaBright-43, VitaBright-48 or monochlorobimane) in WEHI-S cells treated with 10 ng/µL TNF-α. Upper panel (A) shows a schematic drawing of a cytometric scatter plot indicating into which quadrant the different cell populations would fall. Graphs (B-E) are derived from scatter plots and show the percentage of cells present in each quadrant as a function of time of drug treatment. Representative examples of scatter plots can be found in Supporting Information Figure S1a. Nonviable cells were excluded from the scatterplots based on RedDot2 uptake. Data represents the mean ± standard deviation of three independent experiments.

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Figure 5a shows fluorescence intensity histograms of HeLa cells exposed to 0.5 mM hydrogen peroxide for 1–5 h and stained with VitaBright-43, VitaBright-48, monochlorobimane or annexin V CF-647, with nonviable cells excluded based on SYTOX Green staining. The percentage of cells within the marker is shown for each time point. Also for this apoptosis model, the increase in annexin V stained cells correlate with a loss of thiol probe fluorescence intensity, with VitaBright-48 showing the slowest and lowest change.

image

Figure 5. (a) Fluorescence intensities of HeLa cells exposed to 0.5 mM hydrogen peroxide for 1 to 5 hours and stained with VitaBright-43 (VB-43), VitaBright-48 (VB-48) or monochlorobimane (mCB) or annexin V CF-647. The percentage of cells within the marker is shown for each time point. Nonviable cells were gated out based on SYTOX Green uptake. (b) Staining kinetics of the three thiol probes. Histograms show fluorescence intensities of four different cell lines stained with VitaBright-43, VitaBright-48, or monochlorobimane after 1, 3, 5, or 10 min incubation. Nonviable cells were gated out based on PI uptake. (c) Histograms show fluorescence intensities of VitaBright-43, VitaBright-48 or monochlorobimane stained cells treated with either BSO (GSH depleted) or PBS (control). Nonviable cells were excluded based on propidium iodide staining.

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Staining Kinetics

While VitaBright-43 and VitaBright-48 immediately reacts with GSH and other thiols such as dithiothriol in vitro forming fluorescent adducts, monochlorobimane is specific for GSH and will not react with GSH in the absence of glutathione-S-transferase [23]. Figure 5b shows fluorescence intensities of healthy cells, of human as well as rodent origin, stained with VitaBright-43, VitaBright-48 or monochlorobimane after 1, 3, 5, or 10 min incubation. While cells stained with VitaBright-43 and VitaBright-48 immediately (within 1 min) reach maximal fluorescence intensities, the fluorescence intensity of cells stained with monochlorobimane does not reach a plateau within the first 10 min.

Investigation of GSH Specificity

To test the substrate specificity of the three thiol probes HeLa cells were depleted for GSH using the γ glutamylcysteine synthetase inhibitor BSO prior to thiol staining. Figure 5c shows the fluorescence staining intensities of the three thiol probes. The BSO treatment did not affect the fluorescence intensity of VitaBright-48 stained cells compared to the control (PBS treated cells) at the concentration and incubation time used, while the GSH depletion reduced the fluorescence intensity of both VitaBright-43 and monochlorobimane stained cells. Similar results were obtained using other cell lines (not shown). These results strongly indicate that VitaBright-48 reacts with other thiols than GSH.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

An amounting body of evidence links active GSH extrusion with the apoptotic process, and literature documents that the degree of GSH loss, correlates well with the progression of apoptosis [9, 15]. Loss of GSH has been reported to happen in response to a wide range of apoptotic stimuli such as purimycin and etoposide treatment and exposure to FasL [9, 10], and it thus seems to be a preserved characteristic of apoptosis. Changes in the cellular redox level therefore seem to be a marker of apoptosis in line with phospatidylserine externalisation and caspase activity. We aimed to investigate the correlation of reduced thiol level with different early apoptotic markers such as phosphatidylserine externalisation, caspase activity, and loss of the mitochondrial potential in order to explore the possibility of using thiol probes to assay apoptosis. Externalization of phosphatidylserine happens early in the apoptotic process and can be detected by the use of fluorescently tagged annexin V combined with a nonviable stain. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death For measuring caspase activity we have used the Fluorochrome-Labeled Inhibitor of Caspases (FLICA) assay [22]. Another early marker of apoptosis is collapse of the mitochondrial potential. The lipophilic cationic dye JC-1 (5, 5, 6, 6-tetrachloro-1, 1, 3, 3-tetraethylbenzimidazol-carbocyanine iodide) displays potential-dependent accumulation in the mitochondria. Mitochondrial depolarization and hence apoptosis can be observed as a decrease in the red fluorescence intensity of JC-1 stained cells. The three apoptosis assays were chosen to cover an array of very different early apoptotic traits.

A number of different thiol reactive compounds exist, e.g., halo alkyl reagents such as iodoacetamides, which has a high preference for reduced cysteine, but may also target other amino acids in the absence of thiols. Many thiol reactive probes are essentially nonfluorescent, but form fluorescent adducts upon reaction with thiols. The thiol selective halo-bimane monochlorobimane reacts specifically with GSH in a reaction catalyzed by glutathione S-transferase forming a fluorescent monochlorobimane-gluthione conjugate. Monochlorobimane can be used for staining rodent cells; however, it has been reported that glutathione S-transferase present in human cells has a low affinity for monochlorobimane, and GSH is incompletely labeled [24, 25]. Among the thiol reactive probes, probably the most well-explored group are the maleimides, which are far more thiol specific than haloacetamides. Both VitaBright-43 and VitaBright-48 are cell-permeable maleimide derivatives and react with thiols in all cell types by S-alkylation producing fluorescent thioethers independent of glutathione S-transferase activity. Despite VitaBright-43 and VitaBright-48 both are maleimides we show here that they behave differently when used for assaying the level of reduced thiols in cells induced to undergo apoptosis. Thus, VitaBright-43 detects the loss of thiols earlier than VitaBright-48, but at the same time as the GSH specific probe monochlorobimane. Some cells stained intensely with VitaBright-48 are also found to be caspase positive whereas very few cells exhibit this double staining pattern when VitaBright-43 or monochlorobimane is used. This suggests that VitaBright-48 has preference for other cellular components (most likely other thiols) than GSH which are depleted later in the apoptotic process. This difference in GSH specificity is documented in Figure 5c, showing that depletion of GSH using BSO affects staining with VitaBright-43 and monochlorobimane, but not VitaBright-48. Extending the incubation times or increasing the concentration of BSO also reduced fluorescence intensity of viable VitaBright-48 stained cells, however, this also affected cell viability (not shown). VitaBright-43 and monochlorobimane thus appear to be better suited for detection of early apoptosis, while VitaBright-48 may be used as a late apoptosis marker, especially when the very intense and bright staining qualities of this probe are needed. During the initial experimental work we noticed that monochlorobimane often gave inconsistent results with regard to fluorescence intensities even when staining cells from the same cell sample. Exploring this observation we found that timing is crucial for obtaining reproducible results with monochlorobimane. In the presented data, cells stained with monochlorobimane have been incubated at room temperature for exactly 5 minutes, unless otherwise stated (Fig. 5b). Increasing or decreasing the incubation time with as little as one minute changes the measured mean fluorescence intensity and hence the recorded thiol level of the population under investigation. This is in accordance with a study showing that staining of non-rodent cells with monochlorobimane even using prolonged staining (more than 60 minutes) fails to achieve saturation of staining [23]. Moreover, the same study shows that monochlorobimane staining kinetics is highly temperature dependent. Figure 5b shows the changes of fluorescence intensities over time for all three probes for different rodent as well as non-rodent cell lines. Figure 5b documents that the effect of staining kinetics is only important for monochlorobimane, since both VitaBright-43 and VitaBright-48 stained samples immediately reach maximal fluorescence intensity. VitaBright-43 and VitaBright-48 thus provide more reproducible results and are easier to use for detection of changes in cellular thiol level than monochlorobimane due to the staining kinetics. For all three probes we recommend to analyse cells shortly after staining as we noticed increased uptake of non-viable stains (SYTOX Green, RedDot2 and PI) when thiol probe stained cells were incubated more than 30 minutes. This is likely to be a consequence of thiol depletion and may be counteracted by reducing the amount of thiol probe used for staining.

Conventional assays used for determining cellular thiol concentration involve lysis of the cells and measuring the concentration of thiols in the lysate e.g., by HPLC. Thereby information about population dependent distributions is lost. Using cytometric measurement of thiol probe stained cells provide quantification of the thiol level at the single cell level, and allows detection of small changes in the concentration of thiols even of only a subpopulation is affected and can be combined with other markers.

As documented by this study, the decrease in reduced thiols measured using VitaBright-43 or monochlorobimane correlates well with other known assays for early apoptosis. Measuring the thiol level using VitaBright-48 may also be used, but this probe detects changes in the thiol level (and hence apoptosis) later than VitaBright-43 and monochlorobimane. VitaBright-48 stains cells more brightly than VitaBright-43, which again stains cells more intense than monochlorobimane. Moreover, VitaBright-48 is more water soluble and stable than VitaBright-43, however, due to the delayed detection of apoptotic cells, we still recommend to use VitaBright-43 for studies of thiol level changes in correlation to apoptosis, especially in non-rodent cells. As previously mentioned when using either VitaBright-43 or VitaBright-48 the cells immediately reach maximal fluorescence intensity after addition of the probe, while, monochlorobimane needs careful timing. Combined staining with an impermeable stain such as propidium iodide enables exclusion of nonviable cells. As we show here it is also possible to perform multiplexing by adding a third stain e.g. annexin V with a far red fluorescent label. In this case cells should be stained with the annexin V-probe according to manufacturer's recommendations first and then added the thiol probe shortly before analysis. As it is clear from this study all three thiol probes may be used for assaying apoptosis, however, the probes offers different advantages and challenges. Table 1 summarises the pro et contra of the three different probes. All three thiol probes provide a very rapid, easy and reliable way of assaying apoptosis by either flow or image cytometry as no washing or incubation steps are required. Results are also easy to interpret; presenting the data as a histogram showing the fluorescence intensity of all viable cells enables division of cells into subpopulations depending on their thiol level as shown in Figure 5a; apoptotic cells exhibit low fluorescence intensity while healthy cells have a high intensity score. Based on this study we suggest expanding the list of biochemical changes which may be evaluated for detection of apoptosis with measurement of the level of free thiols and recommend VitaBright-43 to be used for this purpose.

Table 1. Summary of advantages and disadvantages of the three thiol probes; VitaBright-43, VitaBright-48 and monochlorobimane
 ProsCons
VB-43Detects early apoptosis Fast and highly reproducible stainingUnknown mechanism for GSH specificity
VB-48Highly fluorescent and reproducible staining of healthy cellsDetects late apoptosis only Not GSH specific
mCBDetects early apoptosis Highly GSH specificSlow and dim staining Staining kinetics cause low reproducibility

Literature Cited

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Literature Cited
  8. Supporting Information

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cytoa22410-sup-0001-suppfig1.docx206KSupporting Information Figure 1.

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