Reduction of MTT to Purple Formazan by Vitamin E Isomers in the Absence of Cells

Abstract

The yellow tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) is widely used to determine cell viability in cell proliferation and cytotoxic assays. MTT is reduced by metabolically active cells to form an insoluble purple formazan product that is quantifiable by spectrophotometry. It is the most common and direct assay for cell viability. However, in this present study, we demonstrated that the vitamin E isomers α-β-γ-δ-tocotrienols and α-tocopherol were able to reduce MTT into a formazan product, despite the absence of living cells. For comparison, a second method for determining cell viability, which is the neutral red uptake assay, was used in parallel with the MTT assay. The results showed that neutral red did not interact with the vitamin E isomers. Our findings suggest that the MTT assay is not suitable for studying the proliferative effects of vitamin E isomers on cell growth.

Keywords: Cell Viability; MTT; Neutral Red Uptake; Vitamin E Isomers.

Figure 1:

MTT results showing the viability percentage of cancerous cells treated with vitamin E isomers, i.e., α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol and α-tocopherol. All cells treated with vitamin E isomers are observed to exhibit reversed cell viability profiles starting at a concentration of 20 µM. Values are presented as the mean percentage ± SD (n = 3).

Figure 2:

Microscopic observations of unstained cancerous cells receiving different treatments: (a) untreated; (b) treated with 10 µM of δ-tocotrienol; (c) treated with 30 µM of δ-tocotrienol; (d) treated with 50 µM of δ-tocotrienol. An increased concentration of δ-tocotrienol inhibits more cancerous cell growth. Bar: 50 µm.

Figure 3:

Reduction of MTT to purple formazans by α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol and α-tocopherol in the absence of cells. A descending ranking order of MTT reduction level can be given as following: γ-tocotrienol > α-tocotrienol > β-tocotrienol > α-tocopherol > δ-tocotrienol. Values are expressed as the mean absorbance ± SD (n = 3).

Figure 4:

Neutral red uptake results showing the viability percentage of cancerous cells treated with α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol and α-tocopherol. Treatment of vitamin E isomers inhibits cancerous cell growth in a concentration-dependent manner. Values are presented as the mean percentage ± SD (n = 3).

Figure 5:

Microscopic observations of neutral red-stained cancerous cells: (a) untreated; (b) treated with 10 µM of δ-tocotrienol; (c) treated with 30 µM of δ-tocotrienol; (d) treated with 50 µM of δ-tocotrienol. Increased concentrations of δ-tocotrienol inhibit more cancerous cell growth. Bar: 10 µm.

Figure 6:

MTT and neutral red uptake results showing the viability percentage of cancerous cells treated with vinblastine serving as a validation control. Treatment of vinblastine inhibits cancerous cell growth in a concentration-dependent manner. Values are presented as the mean percentage ± SD (n = 3).