A Modified MTS Proliferation Assay for Suspended Cells to Avoid the Interference by Hydralazine and b -Mercaptoethanol

The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay is one of the most commonly used tests of cell proliferation. Hydralazine has been reported to interfere with the performance of the MTS assay when used on adherent cells. This study aimed to investigate whether hydralazine interferes with the performance of the MTS assay on suspended cells. THP-1 (a monocytic leukemia cell line) cells were cultured in the presence or absence of hydralazine (0, 10, 50, 100, and 500 l M) for 2 or 24 h. Cell numbers were analyzed using the MTS, trypan blue exclusion, or microscopic assays. A modified version of the standard MTS assay was established by centrifuging the cells and replacing the test medium with fresh culture medium immediately before the addition of the MTS reagent. Culture of THP-1 cells with hydralazine at concentrations of 50, 100, and 500 l M for 2 h increased absorbance (p < 0.001) in the standard MTS assay, whereas both the trypan blue exclusion assay and microscopy suggested no change in cell numbers. Culture of THP-1 cells with 100 and 500 l m hydralazine for 24h increased absorbance (p < 0.05) in the standard MTS assay; however, trypan blue exclusion and microscopy suggested a decrease in cell numbers. In a cell-free system, hydralazine (100 and 500 l M) increased absorbance in a time-and concentration-dependent manner. The modified MTS assay produced results consistent with trypan blue exclusion and microscopy using THP-1 cells. In addition, the modified MTS assay produced reliable results when K562 and Jurkat cells were incubated with hydralazine or b -mercaptoethanol ( b ME). In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and b ME when assessing suspended cells.


INTRODUCTION
I n viable cells, NAD(P)H-dependent oxidoreductase enzymes expressed in mitochondria are capable of converting tetrazolium into colored formazan, [1][2][3][4][5][6][7] proportional to the metabolic activity of mitochondrial enzymes in live cells. The amount of produced formazan can be quantified through absorbance measured by a spectrophotometer and used to estimate the cell numbers. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay relies on this reaction to assay cell numbers. 1 The standard protocol for the MTS assay is very simple. The MTS reagent is directly added to the cultured cells and the absorbance measured after a defined period of incubation.
Hydralazine is an antihypertensive drug that is commonly investigated in heart failure, 8 cancer, 9,10 and blood pressure research. 11 It was recently reported that hydralazine interferes with the performance of MTS assay on adherent cells, 12 and a simple modification of the standard protocol avoids this interference. 12 A number of cell lines are cultured in suspension. The effect of hydralazine on assays of suspended cells has not been previously investigated. This study aimed to examine the effect of hydralazine on the MTS assay of THP-1 (a monocytic leukemia cell line), K562, and Jurkat cells, three commonly used cell lines derived from leukemia. A modified version of the standard MTS assay protocol was developed that included two additional steps, namely centrifuging the suspended cells in a 96-well plate and then aspirating and replacing the test medium with fresh culture medium immediately before the addition of the MTS reagent.
This modified MTS protocol advanced a previously modified protocol 12 of aspirating and replacing the culture medium by adding an extra step of centrifuging the cells. The current protocol overcame the interference with the MTS assay by various testing compounds when suspended cells were used.

MTS Assay
The standard protocol. This was described previously. 12 In brief, cells were seeded at a concentration of 2 · 10 5 cells/mL, 150 mL/well into 96-well flat bottomed tissue culture plates in eight replicates. After the cells were cultured with hydralazine (0, 10, 50, 100, and 500 mM), or b-mercaptoethanol (bME) (0, 100, and 500 mM), or phosphate-buffered saline (PBS) for the specified time, 15 mL of MTS reagent was added into each well and the absorbance was assessed at 490 nm with a microplate reader after 2-h incubation with MTS. 12,14 The modified protocol. This was based upon a recently published modified protocol. 12 In that protocol that was designed for adherent cells, the culture medium in the well was aspirated and replaced with 150 mL fresh, prewarmed standard cell medium immediately before the addition of the 15 mL of MTS reagent. 12 The current modified protocol, which was designed for suspended cells, added an extra step to that protocol, that is, the cells in the 96-well plate were centrifuged at 300 g for 5 min before the culture medium in the well was aspirated.

Trypan Blue Exclusion Assay
About 150 mL of THP-1 (2 · 10 5 /mL) cells were placed in a flatbottom 96-well plate for 24 h. Three microliters of PBS or various concentrations of hydralazine were added to give a final concentration of 0, 10, 50, 100, and 500 mM of hydralazine. Two or 24 h later, 10 mL of cells and 10 mL of trypan blue were mixed and cell numbers were counted using an automated cell counter (Thermo Fisher Scientific, Melbourne, VIC, Australia). 12,14 Imaging Using Light Microscopy Images of the cell culture plates were captured using a contrast phase microscope (Thermo Fisher Scientific). 12

Statistical Analysis
SPSS (version 25) was used for all statistical analyses. Data were presented as meanstandard deviation. Comparisons of mean values were performed by one-way analysis of variance with Bonferroni post hoc test. Differences were considered to be statistically significant at p < 0.05. 12

Overestimate of Live Cell Numbers Using the Standard MTS Assay Protocol in THP-1 Cells
In the cells incubated with hydralazine for 2 h, the results obtained from the standard MTS assay protocol suggested that hydralazine concentration-dependently increased cell numbers (Fig. 1A). Incubation with 50, 100, and 500 mM hydralazine for 2 h appeared to increase the cell number by 2.6, 4.5, and 11.4 times that of the control (0 mM hydralazine) (Fig. 1A). Both the trypan blue exclusion assay (Fig. 1B) and the observations using a light microscope (Fig. 1C) suggested that the cell numbers in each group were similar, suggesting that the standard MTS protocol did not accurately estimate the cell numbers.
Similar findings were obtained from THP-1 cells incubated with hydralazine for 24 h (Fig. 2). The standard MTS assay protocol suggested that incubation with 100 and 500 mM of hydralazine for 24 h increased the cell number to 1.1 and 2.7 times that of the control (0 mM hydralazine) ( Fig. 2A). These findings contradicted the results from the trypan blue exclusion assay (Fig. 2B), which suggested that both 100 and 500 mM hydralazine decreased the live cell numbers. The results from the trypan blue exclusion assay were confirmed by observations using a light microscope (Fig. 2C), which showed that both concentrations of hydralazine (100 and 500 mM) caused cell death.

Direct Reaction of MTS with Hydralazine in a Cell-Free System
The possibility of a direct reaction between MTS and hydralazine was then investigated. In the absence of cells, the absorbance of MTS significantly increased in the presence of hydralazine in a time-and concentration-dependent manner (Fig. 3). This suggested the direct reaction of the colorless MTS with hydralazine to produce a colored compound. The reaction was detectable within 5 min of incubating MTS with hydralazine and reached a plateau around 3 h (Fig. 3).

A Modified MTS Protocol Can Accurately Measure the Live Cell Number of THP-1 Cells Incubated with Hydralazine
The manufacturer's instruction recommends directly adding MTS to the wells containing cells. Given that hydralazine can directly react with MTS, the assay protocol was modified by centrifuging the cells in the 96-well plate at 300 g for 5 min and replacing the used culture medium in the wells with prewarmed fresh culture medium immediately before the addition of MTS. Using the modified MTS assay protocol, the incubation of THP-1 cells with hydralazine for 2 h did not appear to affect the absorbance (Fig. 4A), consistent with the results of the trypan blue exclusion assay (Fig. 1B) and with the observations from the light microscope (Fig. 1C). The re-sults from the modified MTS assay protocol suggested that incubation of THP-1 cells with 10, 50, 100, and 500 mM of hydralazine for 24 h significantly decreased cell numbers in a concentration-dependent manner (Fig. 4B), which was consistent with the results from the trypan blue exclusion assay (Fig. 2B) and the microscopy observations (Fig. 2C).
Modified MTS Protocol Can Accurately Measure the Live Cell Number of K562 and Jurkat Cells Incubated with Hydralazine or bME Another suspended cell line, that is, K562, was used to validate the modified MTS assay protocol (Fig. 5). The  standard MTS assay protocol overestimated cell numbers in the presence of hydralazine (Fig. 5A) or bME (Fig. 5C). The results from the modified MTS assay protocol correctly showed that the cell numbers in each group were similar (Fig. 5B, D). Similar results were obtained in Jurkat cells (Fig. 6).

DISCUSSION
This study reports a simple two-step modification of the standard MTS assay protocol that overcame the interference of hydralazine when assaying suspended cells. As has been  24 h (B). Then the cells in the 96-well plate were centrifuged at 300 g for 5 min and medium was aspirated and replaced with prewarm (37°C) 150 mL culture medium. Fifteen microliters of MTS was then added and absorbance was measured using a plate reader 2 h after the addition of MTS. The absorbance value in the well containing the medium plus MTS in the absence of cells was regarded as background and subtracted from the absorbance of other wells. The absorbance in the control group (0 mM hydralazine) was regarded as 100% (N = 8). Error bars represent standard deviation. Statistical comparisons were performed using one-way ANOVA followed by Bonferroni post hoc tests. previously reported in adherent cells (vascular smooth muscle cells), 12 a discrepancy in the findings of the standard MTS assay protocol and the trypan blue exclusion and microscopic assays was found. A simple modification of the MTS assay protocol, involving centrifuging the suspended cells and replacing the used culture medium immediately before the addition of the MTS reagent was established. The results from the modified MTS assay protocol were in agreement with the results from both the trypan blue exclusion and microscopic assays.
Hydralazine is a reducing reagent that can reduce many oxidizing compounds. 15,16 The current report found that a reaction between hydralazine and the MTS reagent occurred in a time-dependent manner and was completed around 3 h after mixing the two compounds. It was recently reported that the reaction between 100 mM hydralazine and the MTS reagent completed within 2 h. 12 The reaction medium in the Immediately after the addition of hydralazine or bME, 15 mL of MTS was then added to cells in (A, C) (the standard MTS protocol); or the cells in (B, D) were centrifuged at 300 g for 5 min and medium was aspirated and replaced with prewarm 150 mL culture medium followed by addition of 15 mL of MTS (the modified MTS protocol). Absorbance was measured using a plate reader 1.5 h after the addition of MTS. The absorbance value in the well containing the medium plus MTS in the absence of cells was regarded as background and subtracted from the absorbance of other wells. The absorbance in the control group (0 mM hydralazine or 0 mM bME) was regarded as 100% (N = 8). Error bars represent standard deviation. Statistical comparisons were performed using oneway ANOVA followed by Bonferroni post hoc tests. bME, bmercaptoethanol. previous report was culture medium with 10% fetal bovine serum. 12 The reaction medium used in this study was PBS. The quicker reaction rate in the previous report is likely owing to components of fetal bovine serum facilitating the reaction between hydralazine and the MTS reagent.
In the presence of THP-1 cells, the standard MTS assay protocol suggested that incubation with 10-50 mM of hydralazine for 24 h decreased the cell number, whereas incubation with 100-500 mM of hydralazine for 24 h increased cell number. Both the trypan blue exclusion assay and microscopic observations suggested that incubation with 10-500 mM of hydralazine for 24 h decreased cell numbers. The false results of the standard MTS assay at both 100 and 500 mM for 24 h may be because of the inability of the cultured cells to completely absorb and metalize the large amounts of hydralazine within 24 h. However, when hydralazine was used at 10 mM or 50 mM for 24 h, the cells may be able to metabolize the hydralazine, and therefore hydralazine when applying at 10 or 50 mM for 24 h did not interfere with the standard MTS assay.
The results from the modified MTS assay were in good agreement with those from the trypan blue exclusion assay, suggesting that the modified MTS assay is a good alternative method to the trypan blue exclusion assay. The experiment of 24 h preincubation of THP-1 cells with hydralazine showed that the readings of the modified MTS assay were slightly higher (*10%) compared with the trypan blue exclusion assay. This difference could be because of the difference in measurement mechanism: the trypan blue exclusion assay relies on cell membrane integrity, whereas the MTS assay relies on NAD(P)H-dependent oxidoreductase activity inside cells. It is possible that immediately after cells lose their member integrity, a small proportion of the NAD(P)Hdependent oxidoreductase may be still active, resulting in a slightly higher reading in the MTS assay. However, the possibility of a small degree of reversible cytotoxicity of hydralazine during the 2-h incubation with the MTS reagent cannot be excluded.
One limitation of the modified MTS protocol is that it depends on the availability of a plate centrifuge. To avoid inaccurate result, supplementation of the MTS assay with other assays such as the trypan blue exclusion assay, flow cytometry, and microscopic observations is recommended. 17 In addition, this study only tested a limited number of cell lines and interfering compounds. Whether the modified MTS assay applies to other suspended cell lines or other interfering compounds needs to be investigated in the future.
In summary, this study suggests an important limitation of the standard MTS assay protocol when working with suspended cells. A two-step modification of the standard protocol rectifies this problem and results in accurate cell number estimation.

DISCLOSURE STATEMENT
No competing financial interests exist.