Advances in Clinical and Experimental Medicine
2008, vol. 17, nr 5, September-October, p. 525–529
Publication type: original article
Language: English
Limitations of the MTT Assay in Cell Viability Testing
Ograniczenia testu MTT w ocenie żywotności komórek
1 Department of Basic Medical Sciences, Silesian Piasts University of Medicine in Wrocław, Poland
Abstract
Background. The MTT assay is widely recommended for examining the cytotoxic effect of xenobiotics, assessing proliferation rates, and analyzing cell activity. The aim of this study was to evaluate the usefulness of the MTT assay in examining human lymphocyte viability in own cell culture systems which contained fluphenazine (FPh), a suspected cancer chemopreventive agent, and doxorubicin (DOX), an anticancer drug, in cell culture medium.
Material and Methods. Cell culture viability was estimated with two tests: the MTT assay and the propidium iodide (PI) exclusion assay. These results were compared using the standard ANOVA procedure.
Results. The propidium iodide exclusion−test, a microscopic assay of cell culture viability, revealed an increase in dead cell number in cultures treated with DOX and an absence of viable cell changes in cultures treated with FPh in the range of the tested concentrations. The results obtained with the MTT reduction assay in the cultures gave the opposite results; the assay did not mirror the real viable cell number in these cultures. Statistical analysis demonstrated that the results obtained with the two assays differed significantly.
Conclusion. This study shows that the MTT assay is not a feasible tool for determining cell viability in cultures in which DOX and FPh are used and possible sources of the limitations of the MTT assay are discussed.
Streszczenie
Wprowadzenie. Test MTT jest powszechnie zalecany do badania cytotoksycznego działania ksenobiotyków, znajduje także zastosowanie w ocenie proliferacji i aktywności komórek.
Cel pracy. Ocena przydatności testu MTT do analizy żywotności limfocytów w hodowlach zawierających w medium hodowlanym flufenazynę (FPh), potencjalny związek chemoprewencyjny, i doksorubicynę (DOX), przeciwnowotworowy lek cytostatyczny.
Materiał i metody. Ocenę żywotności komórek przeprowadzono w dwóch testach: redukcji MTT i eliminacji jodku propydyny (PI). Wyniki otrzymane w obu testach poddano analizie statystycznej metodą ANOVA.
Wyniki. Wyniki uzyskane w teście spektofotometrycznej oceny redukcji MTT w hodowlach prowadzonych w obecności doksorubicyny oraz w obecności flufenazyny dały wyniki odmienne niż uzyskane w teście oceny mikroskopowej komórek po wybarwieniu hodowli jodkiem propydyny. Analiza statystyczna wykazała, że wyniki otrzymane w obu testach różniły się istotnie.
Wnioski. Przeprowadzone badania pokazały, że testu MTT nie można zastosować do oceny żywotności komórek w hodowlach prowadzonych w obecności DOX i FPh, a ograniczenia zastosowania testu MTT i ich najczęstsze przyczyny są w pracy omawiane.
Key words
MTT assay, PI exclusion−test, lymphocyte cultures, doxorubicin, fluphenazine
Słowa kluczowe
test MTT, test eliminacji PI, hodowle limfocytów, doksorubicyna, flufenazyna
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