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. 2023 Oct;75(5):381-390.
doi: 10.1007/s10616-023-00584-0. Epub 2023 Jul 5.

Assessing MTT and sulforhodamine B cell proliferation assays under multiple oxygen environments

Ming Yao  1   2 , Glenn Walker  3   4 , Michael P Gamcsik  3
Affiliations

Assessing MTT and sulforhodamine B cell proliferation assays under multiple oxygen environments

Ming Yao et al. Cytotechnology. 2023 Oct.

Abstract

Cell proliferation can be measured directly by counting cells or indirectly using assays that quantitate total protein or metabolic activity. However, for comparing cell proliferation under varying oxygen conditions it is not clear that these assays are appropriate surrogates for cell counting as cell metabolism and protein synthesis may vary under different oxygen environments. We used permeable bottom tissue culture ware to compare proliferation assays as a function of static oxygen concentrations under oxygen partial pressure (pO2) levels ranging from 2 to 139 mmHg. Cell proliferation was measured by cell counting and compared to surrogate methods measuring cell metabolism (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) and total protein (sulforhodamine B) assays under these different environments in Caco-2, MCF-7, MCF-10A and PANC-1 human cell lines. We found that the MTT readings do not correlate with cell number for the Caco-2 and PANC-1 cell lines under different oxygen conditions, whereas the sulforhodamine B protein assays perform well under all conditions. However, within a given oxygen environment, both proliferation assays show a correlation with cell number. Therefore, the MTT assay must be used with caution when comparing cell growth or drug response for cells grown in different oxygen environments.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-023-00584-0.

Keywords: Cancer; Hypoxia; MTT assay; Oxygen partial pressure; Proliferation; SRB.

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Conflict of interest statement

Competing interestsThe authors have no relevant financial or non-financial interests to disclose.

Figures

Fig. 1
Fig. 1
Comparison of the effect of pO2 levels on the growth of PANC-1 cells for 72 h in lumox dishes counted manually or automated cell counting on fibronectin-coated PDMS. Data relative to pO2 at 139 (MAN) or 27 mmHg (AUTO)
Fig. 2
Fig. 2
Comparison of the effect of pO2 levels on the growth of A PANC-1 in 96-well plate for 24, 72 h. B Caco-2 cells in 96-well plate for 24 and 72 h. C MCF-7 cells in 96-well plate for 24, 72 h and sealed bags for 24 h D MCF10A cells in 96-well plates for, 24, 48 and 72 h. All counts and statistical significance (if any) are relative to the data at pO2 = 139 mmHg at the same exposure time. Some data is combined from two trials (× 2) at the given time period
Fig. 3
Fig. 3
Comparison of the effect of pO2 levels on the MTT absorbances of A PANC1 B Caco-2, C MCF-7 and D MCF-10A cultured in fibronectin-coated PDMS 96 well plates. Some data are combined from multiple trials (× n). Symbols indicate statistically significant differences relative to the data at pO2 = 139 mmHg at the same exposure time
Fig. 4
Fig. 4
Relative MTT Absorbances versus cell seeding density for A PANC-1 B Caco-2 C MCF-7 D MCF10A cells cultured for 24 h. The pO2 values are given for each row of data. The lines are the best fit to the data
Fig. 5
Fig. 5
Relative SRB Absorbances versus cell seeding density for A PANC1 and B Caco-2 C MCF-7 and D MCF10A cells. The MCF7 cells were seeded at a 10 K and 20 K (DS) in the 96-well plate. Symbols indicate statistically significant differences relative to the data at pO2 = 139 mmHg at the same exposure time
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References

    1. Adan A, Kiraz Y, Baran Y. Cell proliferation and cytotoxicity assays. Curr Pharm Biotechnol. 2016;17:1213. doi: 10.2174/1389201017666160808160513. - DOI - PubMed
    1. Ahmadi M, Ahmadihosseini Z, Allison SJ, Begum S, Rockley K, Sadiq M, Chintamaneni S, Lokwani R, Hughes N, Phillips RM. Hypoxia modulates the activity of a series of clinically approved tyrosine kinase inhibitors. Br J Pharmacol. 2014;171:224. doi: 10.1111/bph.12438. - DOI - PMC - PubMed
    1. Al-Ani A, Toms D, Kondro D, Thundathil J, Yu Y, Ungrin M. Oxygenation in cell culture: critical parameters for reproducibility are routinely not reported. PLoS ONE. 2018;13:e0204269. doi: 10.1371/journal.pone.0204269. - DOI - PMC - PubMed
    1. Angius F, Floris A. Liposomes and MTT cell viability assay: an incompatible affair. Toxicol in Vitro. 2015;29:314. doi: 10.1016/j.tiv.2014.11.009. - DOI - PubMed
    1. Bagshaw ORM, De Lange M, Renda S, Valente AJF, Stuart JA. Hypoxio: a simple solution to preventing pericellular hypoxia in cell monolayers growing at physiological oxygen levels. Cytotechnology. 2019;71:873. doi: 10.1007/s10616-019-00326-1. - DOI - PMC - PubMed

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