The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts

Abstract

In the past years, several methods have been developed to determine cell viability in cell culture (Cook and Mitchell, 1989). Among these methods tetrazolium salt-based assays are widely used in order to measure cytotoxicity or cell proliferation (Mosmann, 1983;Berridge et al., 1996).The principle of the MTT [3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay is the reduction of water-soluble yellow tetrazolium salt by the dehydrogenase system of metabolically active/live cells into water-insoluble blue/magenta (MTT) formazan crystals (Morgan et al., 1998). In this way, the concentration of dissolved formazan crystals can be quantified using a spectrophotometer and it is in direct correlation to the number of metabolically active cells (Gabrielson et al., 2002;Tunney et al., 2004;Wang et al., 2010). The MTT assay represents a simple and rapid colorimetric assay and yields quantitative data (Alley et al., 1988). This assay is carried out entirely in 96-well microtiter plates; thus, large numbers of experiments examining a number of variables can be readily performed (Cole, 1986).On the other hand, the MTT assay has some disadvantages that are dependent on the cell ability to overcome cell death. One remarkable disadvantage is that damaged mitochondria may be still able to reduce MTT to formazan crystals (Mosmann, 1983;Page et al., 1988;Sieuwerts et al., 1995). Loveland et al. (1992) showed that cells with inactivated mitochondria were also able to produce formazan crystals as well as cells with active mitochondria. Furthermore, many nonmitochondrial dehydrogenases and flavin oxidases are able to reduce MTT (Altman, 1976;Burdon et al., 1993). Besides, different conditions and some chemicals/phytochemicals can also lead to changes in metabolic activity (Plumb et al., 1989;Hsu et al., 2003). The MTT compound may interact with some chemicals/phytochemicals, resulting in false results in viability (

Transepithelial Anti-Neuroblastoma Response to Kale among Four Vegetable Juices Using In Vitro Model Co-Culture System

“…Bernhard et al [ 62 ] first demonstrated this phenomenon using the CEM-C7H2 lymphocytic leukemia cell line during uptake of resveratrol which chemically reduced MTT molecules inside the cells in the face of declining cell growth. Similar findings have been documented repeatedly with plant extracts to the point where we now consider this MTT phenomenon a signature of such molecules [ 64 ]. Hence, when observed that basolateral SH-SY5Y cells in our co-cultures had declining cell numbers in the face of sustained high MTT metabolism ( Figure 3 ), it most likely means that antioxidant flavonoids from kale juice had traversed the Caco-2Bbe1 barrier and entered these cells.…”

“…Hence, to convert MTT to a purple color theoretically would require more mitochondria—which by adding kale juice seems highly unlikely. A subset of the literature shows, however, that purified flavonoids lead to an “artifactual” reduction of MTT dye [ 61 , 62 , 63 , 64 ]. To observe a full comparison of this experiment in the Caco-2Bbe1 cells, please see Supplementary Figure S1 (less dichotomous).…”

“…This rank order matched our original hypothesis, which came from the literature thus helping to validate our new coculture model system. That kale’s phytochemicals, themselves, actually cross the Caco-2Bbe1 barrier seems evidenced by the neuroblastoma cells showing a signature MTT change (discussed later about MTT formazan dye reduction), well known for flavonoids [ 62 , 63 , 64 ].…”

“…Reduction of yellow MTT to a purple formazan by enzymes supposedly confined in mitochondria is widely considered to measure cell health, and therefore the number of cells in a dish [ 61 ]. However, the MTT method is also known for a signature effect when uncoupled by flavonoid phytochemicals [ 62 , 63 , 64 ]. To show this, our cells were washed with PBS (0.5 mL) to separately collect (for later) the floaters.…”

Inhibition of Growth and Migration of Esophageal Squamous Cell Carcinoma Cells by Orange Peel Extract and Naringin

“…The cells were cultured in Dulbecco's modified Eagle's medium: F12 (DMEM: F12) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 5% CO 2 and 37 C under standard conditions. Cell viability was assessed using MTT assay with slight modifications (16).…”