Anal Biochem. 2016 Sep 15;509:33-40.
Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays
Jennifer Jin Ruana, Benfang Helen Ruanb* et al
a Hangzhou Jennifer Biotech. Inc., Hangzhou, China
b College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, China * Corresponding author. Current address: 18 Chaowang Road, Xiachengqu, Hangzhou, Zhejiang, 310014, China E-mail addresses: firstname.lastname@example.org, email@example.com (B.H. Ruan).
Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H2O2 oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC50 45 nM) and found that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC50 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% inhibition was observed for WST- 8. In conclusion, EZMTT is a novel tetrazolium reagent which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).
KEYWORDS: Cell proliferation; Cell toxicity; Dehydrogenase; High through-put screening; Mono-sulfonated tetrazolium salt; NAD(P)H assay
Oxidoreductases are popular drug targets in pharmaceutical research, and their cofactors NAD+/NADH and NAP+/NADPH are involved in various important cellular process, such as energy metabolism, mitochondrial function, oxidative stress, calcium homeostasis, antioxidation, generation of oxidative stress, gene expression, immunological functions, and cell death . For example, NAD and NADP regulate key factors that are involved in cell death; the key factors include mitochondrial permeability transition (MPT), poly(ADP-ribose) polymerase-1 (PARP-1) and apoptosis-inducing factors.
The MPT is a mediator of apoptosis that is greatly affected by the NAD/NADH ratio. Also MPT mediates the PARP-1 activation-induced mitochondrial NAD+ loss, which contributes to metabolic dysfunction. PARP-1 inhibits the catalytic activities of the protein for Werner’s syndrome and causes NAD reduction that leads to decreased mitochondrial activity and premature aging.
In addition, increasing evidence has suggested that NADP could significantly contribute to generation of oxidative stress through the activity of NADPH oxidase. NADH and NADPH depletion are events that happen early in the cell death process. NAD mediates the mitochondrial energy metabolism, which makes it a critical factor in determining cell death. NADH is one of the major electron donors for the electron transport chain that leads to the most significant ATP synthesis. Therefore, NAD+ is the coenzyme for these rate-limiting reactions that are involved in the ATP synthesis through the TCA cycle.
Owing to these critical roles, detection of NADH/NADPH in cells is becoming an increasingly useful tool in cancer research. Current detection methods for NADH and NAD(P)H utilize UV spectroscopy (350 nm) and the tetrazolium-formazan-NAD(P)H method. As shown in Figure 1A, UV spectroscopy measurement at 340 nm showed poor sensitivity and gave low signal to background ratio. WST-8 (or CCK-8; Figure 1B) is a tetrazolium reagent, which provided at least 10-fold higher sensitivity in NAD(P)H detection. However, WST-8 can be reduced not only by NAD(P)H but also other antioxidants, such as the thio-group containing compound BME, which can cause false positives during the assay. EZMTT is a newly developed mono-sulfonated tetrazolium salt, designed to react with NAD(P)H, but not (or very weakly) with other antioxidants (Figure 1C).
Figure 1. NAD(P)H assay. A) NAD(P)H assay by UV absorbance at 340 nm; B) NAD(P)H measured by continuous WST-8 or EZMTT assays; C) Reaction of WST-8 or EZMTT with antioxidant BME; D) EZMTT Detection Method: a single step addition of a 5X solution to the cell culture, and then the cell viability is determined by UV absorbance at 450nm after a one-hour incubation time. The maximum number of the cells will have the darkest orange color; E) The linear dose response curve for the cell numbers; F) Z factor measurement
EZMTT is found to be a robust, reliable, affordable, and easy to use method for rapid continuous analyses, such as NADH and NAD(P)H quantification, dehydrogenase activity assay, and real time live cell-viability assay. Figure 1D depicted the process of the one step EZMTT cell viability assay: simply dilute the reagents in culture media, add the resulting 5X EZMTTworking solution to each well, grow the cells in a 37︒C incubator for 1-4 hours, and read the plate at UV absorbance of 450 nm for cell viability. The assay provides a low background value in the absence of cells and has a linear relationship between cell number and signal produced (Figure 1E); this is essential for accurate IC50 values and an excellent reproducibility in IC50 measurement has been achieved with a Z factor of 0.7 (Figure 1F) .
Currently, the most popular commercially available methods for cell viability assay are the ATP-based Cell-Titer Glo and the NAD(P)H based WST-8 and MTT Assays. Because NAD(P)H is the major electron donor for the electron transport chain that leads to the ATP synthesis in mitochondia, these assays are expected give essentially the same results in cell viability assays. To compare EZMTT with these 3 assay formats, we performed the IC50 measurement of 4 compounds (HJ7, CB-839, Doxorubicin, and HJ89) in two cancer cell lines (CaKi and A375 cells;104 cells/well) using RPMI 1640 with 10% FBS serum. The changes in IC50 values over a time course of 24 hours were followed, after addition of individual detection reagents (Cell Titer Glo, MTT, EZMTT, and WST-8).
The CellTiter-Glo Luminescent Cell Viability Assay  determines the number of viable cells by measuring the ATP levels; in the presence of ATP and oxygen, the thermostable luciferase (Ultra-Glo Recombinant Luciferase) converts luciferin into oxyluciferin which emits light. The assay is rapid, and the strong luminescent signal can be observed in 30min, then signal started to drop and totally disappeared in 24 hrs, as shown in Figure 2A. In addition, IC50 values changed with time, and for some compounds, the percentage of inhibition did not reach 0% at the early assay time points (10 min – 1hr) ,which causes problems in accuracy of IC50 determination (Figure 2B, 2C, 2D). Also, since the chemiluminescence formation is catalyzed by Luciferase, compounds that inhibit luciferase might give false positives. Also, the assay requires use of an opaque assay plate that prevents periodic microscopic observation of cell morphological changes during the drug treatment.
Figure 2. Cell viability assay: Cancer cells (A375 or Caki-1; 1000) were plated on to a 96 well plate (Opaque plate for Cell Titer Glo assay; clear bottom plate for MTT, WST-8, EZMTT assays), grown for 4 hrs later, and then treated with compounds (0 – 10 µM) in triplicate for 5 days. HJ7 is a KGA/GDH dual inhibitor, CB-839 a KGA inhibitor, Doxorubicin, and HJ89 a BPTES type KGA inhibitor. The cell viability was determined by four different assays with the following procedure: 1. directly add ATP based Cell Titer Glo reagents (1:1) and 10 min later measure its chemiluminescence. A) Signal changes at different time points (0.5, 1, 2, 3 hrs); B) IC50 determination for DOX inhibitor at different time points; C) IC50 determination for HJ7 inhibitor at different time points; D) IC50 determination for HJ89 inhibitor at different time points; 2. add NAD(P)H based MTT reagents for 3-4 hours, remove the culture media, and add DMSO to solubilize the purple formazan for measurement of UV absorbance at 570 nm: E) Signal changes at different time points (0.5, 1, 2, 3 hrs); F) IC50 for DOX; G) IC50 for HJ7; H) IC50 for HJ89; 3. add NAD(P)H based WST-8 assay and 30 min later measure its UV absorbance at 450 nm : I) Signal changes at different time points (0.5-12 hrs); J) IC50 for DOX; K) IC50 forHJ7; L) IC50 for HJ89; 4. add NAD(P)H based EZMTT assay, and 30 min later measure its UV absorbance at 450 nm. M) Signal changes at different time points (0.5-12 hrs). N) IC50 for DOX; O) IC50 forHJ7; P) IC50 for HJ89.
The second assay is the MTT Cell Proliferation assay, which is the traditional method for NAD(P)H determination. The yellow tetrazolium MTT is reduced to the purple-colored formazan by NAD(P)H that is constantly generated in metabolically active cells through the action of dehydrogenase enzymes. This assay requires 3-4 hrs incubation time with tetrazolium MTT in order to get significant intensity of signal, and an additional step of DMSO solubilization is necessary for its water insoluble formazan quantification. In general, the MTT and the CellTiter-Glo assay both are end point assays, but MTT requires at least 2 steps of reagent additions and takes significant longer experiment times. However, a clear bottom plate can be used for the MTT assay because the read out is the UV absorbance at 540 nm (Figure 2E-H).
The WST-8 (CCK8) Cell Proliferation Assay Kit uses a soluble formazan that provides several advantages over the traditional MTT assay. First, since the corresponding formazan of WST-8 is water soluble, the DMSO step is not required. Therefore the WST-8 assay requires only a single step of reagent addition and provides a continuous assay of NAD(P)H level. However, in our experiments, we discovered that its main flaw is the false positive signal when antioxidant inhibitors were tested; WST-8 can be reduced not only by NAD(P)H, but also by various antioxidants generating the yellow-colored water soluble formazan that is detected at UV absorbance of 350 nm. Further, to our disappointment, we found that even though WST-8 provides a continuous assay over a period of 24 hours (Figure 2I), the IC50 values change with time (Figure 2J-2L). This significantly reduces reproducibility in IC50 determination, so the assay is not suitable for high throughput screening.
The EZMTT reagent kit provides a continuous assay like the WST-8 assay (Figure 2M), and importantly essentially the same IC50 values as those measured by other detection methods (Table 1). To our surprise, the EZMTT assay demonstrated essentially identical IC50 curves over a period of 12 hours (Figure 2N-2P), whereas the other methods show several-fold variations in IC50 measurement over 12 hours (Figure 2A-2L, Table 1). The data clearly demonstrate that EZMTT could replace the other assays for cell viability. In addition, the consistent IC50 values over a period of 12 hrs is a highly desirable feature for high throughput screening.
Table 1. IC50 measurement of cancer cell growth inhibition by four different commercially available cell viability assay kits
1Optimal IC50 measured at 30 minutes by Cell Titer Glo assay; Range, IC50 measured 0.5, 1, 2, 3, 4 hours.
2Optimal IC50 measured at 2 hours by EZMTT assay; Range, IC50 measured 0.5, 1, 2, 3, 4, 6,12 hours
3Optimal IC50 measured at 3 hours by MTT assay; Range, IC50 measured 3, 4, 6,12 hours
4Optimal IC50 measured at 1 hour by WST-8 assay; Range, IC50 measured 0.5, 1, 2, 3, 4, 6,12 hours
In addition, the EZMTT reagent demonstrated the great advantage of being non-cytotoxic to the cells; in cell toxicity assay (Figure 3A), EZMTT showed less than 5% of growth inhibition, while WST-8 showed 40% growth inhibition under the same conditions. For further validation, the cells used in the EZMTT or WST-8 assays were washed with PBS, and the washed cells were treated with Cell Titer Glo reagents. Notably, the Cell Titer Glo assay showed that the EZMTT pretreatment caused much less of a shift in the IC50 curve than that of the WST-8 pretreatment, in comparison to the non-pretreated cells (figure 3B).
Figure 3. The toxicity of EZMTT versus WST-8: A) IC50 measurement. At 0.5 mM concentration, EZMTT showed less than 5% growth inhibition, whereas WST-8 showed 40% inhibition B) Cell Titer Glo assay: Three sets of A375 cancer cells were treated with DOX under the same condition, and the growth inhibitions were measured by Cell Titer Glo, WST-8 and EZMTT assays. The cells that had been treated with WST-8 or EZMTT reagents for 24 hrs were washed with PBS twice and then added Cell Titer Glo reagents for IC50 measurements as presented in the Figure. EZMTTpretreatment for 24 hrs had minimal effect on IC50 measurement using Cell Titer Glo assay.
In conclusion, EZMTT is a newly found water-soluble tetrazolium reagent suitable for cell viability assays. In comparison with several popular cell viability assay formats such CellTiter Glo, WST-8, and MTT, EZMTT provided similar accuracy in IC50 measurement. In addition, the EZMTT assay is a continuous assay demonstrating essentially identical IC50 values over a period of nearly 10 hours which is highly desirable for high throughput screening. Also EZMTT is stable under storage at -20 °C, has low toxicity, causes minimal false positive reactions with antioxidants, and provides the scientific community with moderately priced reagents for daily routine cancer drug screening.
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