A modified MTS proliferation assay for suspended cells to avoid the interference by Hydralazine and beta-Mercaptoethanol

Wang, Yutang, Nguyen, Dinh Tam, Yang, Guang, Anesi, Jack, Kelly, Jason, Chai, Zhonglin, Ahmady, Fahima, Charchar, Fadi, and Golledge, Jonathan (2021) A modified MTS proliferation assay for suspended cells to avoid the interference by Hydralazine and beta-Mercaptoethanol. Assay and Drug Development Technologies. (In Press)

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View at Publisher Website: https://doi.org/10.1089/adt.2020.1027
 
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Abstract

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 mu 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 mu 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 mu m 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 mu 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 beta-mercaptoethanol (beta ME). In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and beta ME when assessing suspended cells.

Item ID: 66060
Item Type: Article (Research - C1)
ISSN: 1540-658X
Keywords: cytotoxicity, hydralazine, MTS proliferation assay, suspension cells
Copyright Information: Copyright 2021, Mary Ann Liebert, Inc., publishers
Funders: National Health and Medical Research Council of Australia (NHMRC), Queensland Government (QG)
Projects and Grants: NHMRC grant 1062671, NHMRC Practitioner Fellowship 1117061, QG Senior Clinical Research Fellowship
Date Deposited: 17 Feb 2021 18:02
FoR Codes: 32 BIOMEDICAL AND CLINICAL SCIENCES > 3201 Cardiovascular medicine and haematology > 320199 Cardiovascular medicine and haematology not elsewhere classified @ 100%
SEO Codes: 20 HEALTH > 2001 Clinical health > 200105 Treatment of human diseases and conditions @ 100%
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