Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS

Abstract

Reported parameters of the MTT assay vary widely, and reflect a need to optimise the assay for different cell types. The MTT assay conditions for the human B-lymphocyte-derived cell line WIL2NS were optimised for MTT incubation and formazan development. The optimised MTT assay was validated by examining the effects of the acaride amitraz on WIL2NS. In pH-buffered media in the absence of cells, MTT formed formazan spontaneously, and absorbance was proportional to both the initial concentration of MTT and the time of incubation at 37°C. One milligram per millilitre MTT was toxic to WIL2NS cells, but the accuracy of the standard curve was reduced when only 0.2 mg/ml MTT was used. Twenty percent SDS in 0.2 M HCl was preferable to DMSO as a solvent for formazan. Exposure to 0.035% amitraz resulted in a significant reduction in WIL2NS cell numbers after only 2 h of exposure. It was concluded that 0.035% of amitraz has the potential to adversely affect lymphocytes in the systemic blood system in humans, and that an optimised MTT assay was obtained by incubating WIL2NS cells with 0.45 mg/ml MTT for 17 h, followed by addition of acidified SDS for 1 h. © 2005 Elsevier Ltd. All rights reserved.