Articles

Article

Original Article

Exp Neurobiol 2015; 24(3): 235-245

Published online September 30, 2015

https://doi.org/10.5607/en.2015.24.3.235

© The Korean Society for Brain and Neural Sciences

The Unreliability of MTT Assay in the Cytotoxic Test of Primary Cultured Glioblastoma Cells

Hwa Yeon Jo1,2,3, Yona Kim1,2,3, Hyung Woo Park1,2,3, Hyo Eun Moon1,2,3, Seongtae Bae1,2,3, JinWook Kim1,2,3, Dong Gyu Kim1,2,3 and Sun Ha Paek1,2,3*

1Department of Neurosurgery, 2Cancer Research Institute, and 3Ischemic/Hypoxic Disease Institute,Seoul National University College of Medicine, Seoul 03082, Korea

Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-2072-3993, FAX: 82-2-744-8459
e-mail: paeksh@snu.ac.kr

Received: August 20, 2015; Revised: August 30, 2015; Accepted: August 31, 2015

Abstract

MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.

Keywords: primary cell, ethanol, Tetrazolium salts, non-specific intracellular reduction