An Improved 3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2H-Tetrazolium Proliferation Assay to Overcome the Interference of Hydralazine

Wang, Yutang; Nguyen, Dinh Tam; Guang, Yang; Anesi, Jack; Chai, Zhonglin; Charchar, Fadi J.; Golledge, Jonathan

doi:10.1089/adt.2020.1004

Cited by 8 publications

(19 citation statements)

References 17 publications

(20 reference statements)

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“…The THP-1, K562, and Jurkat cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640) or Dulbecco's modified Eagle's medium as previously described. 12,13…”

Section: Cell Culturementioning

confidence: 99%

“…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.…”

Section: Mts Assaymentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Wang

Nguyen

Guang

et al. 2021

ASSAY and Drug Development Technologies

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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 lM) 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 lM 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 lm hydralazine for 24 h 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 lM) increased absorbance in a time-and concentrationdependent 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 (bME). In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and bME when assessing suspended cells.

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“…The THP-1, K562, and Jurkat cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640) or Dulbecco's modified Eagle's medium as previously described. 12,13…”

Section: Cell Culturementioning

confidence: 99%

Section: Mts Assaymentioning

confidence: 99%