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.
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References
Adan A, Kiraz Y, Baran Y (2016) Cell proliferation and cytotoxicity assays. Curr Pharm Biotechnol 17:1213
Ahmadi M, Ahmadihosseini Z, Allison SJ, Begum S, Rockley K, Sadiq M, Chintamaneni S, Lokwani R, Hughes N, Phillips RM (2014) Hypoxia modulates the activity of a series of clinically approved tyrosine kinase inhibitors. Br J Pharmacol 171:224. https://doi.org/10.1111/bph.12438
Al-Ani A, Toms D, Kondro D, Thundathil J, Yu Y, Ungrin M (2018) Oxygenation in cell culture: critical parameters for reproducibility are routinely not reported. PLoS ONE 13:e0204269. https://doi.org/10.1371/journal.pone.0204269
Angius F, Floris A (2015) Liposomes and MTT cell viability assay: an incompatible affair. Toxicol in Vitro 29:314. https://doi.org/10.1016/j.tiv.2014.11.009
Bagshaw ORM, De Lange M, Renda S, Valente AJF, Stuart JA (2019) Hypoxio: a simple solution to preventing pericellular hypoxia in cell monolayers growing at physiological oxygen levels. Cytotechnology 71:873. https://doi.org/10.1007/s10616-019-00326-1
Bakmiwewa SM, Heng B, Guillemin GJ, Ball HJ, Hunt NH (2015) An effective, low-cost method for achieving and maintaining hypoxia during cell culture studies. Biotechniques 59:223. https://doi.org/10.2144/000114341
Carreau A, El Hafny-Rahbi B, Matejuk A, Grillon C, Kieda C (2011) Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med 15:1239. https://doi.org/10.1111/j.1582-4934.2011.01258.x
Costanzini A, Sgarbi G, Maresca A, Del Dotto V, Solaini G, Baracca A (2019) Mitochondrial mass assessment in a selected cell line under different metabolic conditions. Cells 8:E1454. https://doi.org/10.3390/cells8111454
Funk D, Schrenk HH, Frei E (2007) Serum albumin leads to false-positive results in the XTT and the MTT assay. Biotechniques 43:180
Jensen MD, Wallach DFH, Sherwood P (1976) Diffusion in tissue cultures on gas-permeable and impermeable supports. J Theoret Biol 56:443
Kumar N, Afjei R, Massoud TF, Paulmurugan R (2018a) Comparison of cell-based assays to quantify treatment effects of anticancer drugs identifies a new application for Bodipy-L-cystine to measure apoptosis. Sci Rep 8:16363. https://doi.org/10.1038/s41598-018-34696-x
Kumar P, Nagarajan A, Uchil PD (2018b) Analysis of cell viability by the MTT assay. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot095505
Kupcsik L (2011) Estimation of cell number based on metabolic activity: the MTT reduction assay. Methods Mol Biol 740:13. https://doi.org/10.1007/978-1-61779-108-6_3
Lefebvre VH, Van Steenbrugge M, Beckers V, Roberfroid M, Buc-Calderon P (1993) Adenine nucleotides and inhibition of protein synthesis in isolated hepatocytes incubated under different pO2 levels. Arch Biochem Biophys 304:322. https://doi.org/10.1006/abbi.1993.1357
Lundholt BK, Scudder KM, Pagliaro L (2003) A simple technique for reducing edge effect in cell-based assays. J Biomol Screen 8:566. https://doi.org/10.1177/1087057103256465
Minoves M, Morand J, Perriot F, Chatard M, Gonthier B, Lemarie E, Menut JB, Polak J, Pepin JL, Godin-Ribuot D, Briancon-Marjollet A (2017) An innovative intermittent hypoxia model for cell cultures allowing fast Po2 oscillations with minimal gas consumption. Am J Physiol Cell Physiol 313:C460. https://doi.org/10.1152/ajpcell.00098.2017
Orellana EA, Kasinski AL (2016) Sulforhodamine B (SRB) assay in cell culture to investigate cell proliferation. Bio Protoc 6:e1984. https://doi.org/10.21769/BioProtoc.1984
Place TL, Domann FE, Case AJ (2017) Limitations of oxygen delivery to cells in culture: an underappreciated problem in basic and translational research. Free Radic Biol Med 113:311. https://doi.org/10.1016/j.freeradbiomed.2017.10.003
Polak J, Studer-Rabeler K, McHugh H, Hussain MA, Shimoda LA (2015) System for exposing cultured cells to intermittent hypoxia utilizing gas permeable cultureware. Gen Physiol Biophys 34:235. https://doi.org/10.4149/gpb_2014043
Rexen P, Kierulff JV, Emborg C (1992) An easy-to-handle semi-automated method for media development using a colorimetric viability assay and fractional factorial design. Cytotechnology 8:195
Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, Minor L (2013) Cell viability assays. Assay guidance manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda
Rubinstein LV, Shoemaker RH, Paull KD, Simon RM, Tosini S, Skehan P, Scudiero DA, Monks A, Boyd MR (1990) Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J Natl Cancer Inst 82:1113. https://doi.org/10.1093/jnci/82.13.1113
Schneider CA, Rasband WS, Elcieri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671
Shoemaker RH (2006) The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer 6:813. https://doi.org/10.1038/nrc1951
Stepanenko AA, Dmitrenko VV (2015) Pitfalls of the MTT assay: direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene 574:193. https://doi.org/10.1016/j.gene.2015.08.009
Szmelter A, Jacob J, Eddington DT (2021) 96-well oxygen control using a 3D-printed device. Anal Chem 93:2570. https://doi.org/10.1021/acs.analchem.0c04627
van Tonder A, Joubert AM, Cromarty AD (2015) Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Res Notes 8:47. https://doi.org/10.1186/s13104-015-1000-8
Vaupel P, Mayer A (2007) Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 26:225. https://doi.org/10.1007/s10555-007-9055-1
Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1:1112. https://doi.org/10.1038/nprot.2006.179
Wang S, Yu H, Wickliffe JK (2011) Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2. Toxicol in Vitro 25:2147. https://doi.org/10.1016/j.tiv.2011.07.007
Wenger RH, Kurtcuoglu V, Scholz CC, Marti HH, Hoogewijs D (2015) Frequently asked questions in hypoxia research. Hypoxia (auckl) 3:35. https://doi.org/10.2147/hp.s92198
Yao M, Sattler T, Rabbani ZN, Pulliam T, Walker G, Gamcsik MP (2018) Mixing and delivery of multiple controlled oxygen environments to a single multiwell culture plate. Am J Physiol Cell Physiol 315:C766
Yao M, Rabbani ZN, Sattler T, Nguyen KG, Zaharoff D, Walker G, Gamcsik M (2019) Flow-encoded oxygen control to track the time-dependence of molecular changes induced by static or cycling hypoxia. Anal Chem 91:15032. https://doi.org/10.1021/acs.analchem.9b03709
Yao M, Walker G, Gamcsik MP (2021) A multiwell plate-based system for toxicity screening under multiple static or cycling oxygen environments. Sci Rep 11:4020. https://doi.org/10.1038/s41598-021-83579-1
Zeitouni NE, Fandrey J, Naim HY, von Kockritz-Blickwede M (2015) Measuring oxygen levels in Caco-2 cultures. Hypoxia (aucKl) 3:53. https://doi.org/10.2147/hp.S85625
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This work was supported by the National Institutes of Health (Grant Number: R21CA202804).
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All authors contributed to the study conception and design. Data collection was performed by MY. Data analysis was performed by MY and MPG. The first draft of the manuscript was written by MPG and reviewed and edited by MY and GW. All authors read and approved the final manuscript.
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Yao, M., Walker, G. & Gamcsik, M.P. Assessing MTT and sulforhodamine B cell proliferation assays under multiple oxygen environments. Cytotechnology 75, 381–390 (2023). https://doi.org/10.1007/s10616-023-00584-0
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DOI: https://doi.org/10.1007/s10616-023-00584-0