The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity....

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Veröffentlicht in:	International journal of molecular sciences Jg. 22; H. 23; S. 12827
Hauptverfasser:	Ghasemi, Mahshid, Turnbull, Tyron, Sebastian, Sonia, Kempson, Ivan
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	Switzerland MDPI AG 26.11.2021 MDPI
Schlagworte:	Cell Count Cell Survival Culture Media - pharmacology Cytotoxicity Enzymes Gold - chemistry Humans Male Metabolism Metal Nanoparticles - administration & dosage Metal Nanoparticles - chemistry Polyethylene glycol Prostatic Neoplasms - drug therapy Prostatic Neoplasms - metabolism Prostatic Neoplasms - pathology Reagents Secretome Single-Cell Analysis - methods Tumor Cells, Cultured how to gold nanoparticles PC-3 cells interpret cell metabolism image cytometry MTT assay cytotoxicity viability assay
ISSN:	1422-0067, 1661-6596, 1422-0067
Online-Zugang:	Volltext
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Abstract	The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.
AbstractList	The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations. The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.
Author	Kempson, Ivan Ghasemi, Mahshid Turnbull, Tyron Sebastian, Sonia
AuthorAffiliation	Future Industries Institute, University of South Australia, Adelaide, SA 5095, Australia; mahshid.ghasemi_esfidvajani@mymail.unisa.edu.au (M.G.); Tyron.Turnbull@unisa.edu.au (T.T.); sonia.sebastian@mymail.unisa.edu.au (S.S.)
AuthorAffiliation_xml	– name: Future Industries Institute, University of South Australia, Adelaide, SA 5095, Australia; mahshid.ghasemi_esfidvajani@mymail.unisa.edu.au (M.G.); Tyron.Turnbull@unisa.edu.au (T.T.); sonia.sebastian@mymail.unisa.edu.au (S.S.)
Author_xml	– sequence: 1 givenname: Mahshid surname: Ghasemi fullname: Ghasemi, Mahshid – sequence: 2 givenname: Tyron surname: Turnbull fullname: Turnbull, Tyron – sequence: 3 givenname: Sonia surname: Sebastian fullname: Sebastian, Sonia – sequence: 4 givenname: Ivan orcidid: 0000-0002-3886-9516 surname: Kempson fullname: Kempson, Ivan
BackLink	https://www.ncbi.nlm.nih.gov/pubmed/34884632$$D View this record in MEDLINE/PubMed
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Keywords	how to gold nanoparticles PC-3 cells interpret cell metabolism image cytometry MTT assay cytotoxicity viability assay
Language	English
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Snippet	The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We...
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StartPage	12827
SubjectTerms	Cell Count Cell Survival Culture Media - pharmacology Cytotoxicity Enzymes Gold - chemistry Humans Male Metabolism Metal Nanoparticles - administration & dosage Metal Nanoparticles - chemistry Polyethylene glycol Prostatic Neoplasms - drug therapy Prostatic Neoplasms - metabolism Prostatic Neoplasms - pathology Reagents Secretome Single-Cell Analysis - methods Tumor Cells, Cultured
Title	The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis
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