Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS.
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 degrees 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.
Young
Fiona M
FM
Department of Medical Biotechnology, School of Medicine, Flinders University of South Australia, Bedford Park, SA 5042, Australia.
Phungtamdet
Wichaya
W
Sanderson
Barbara J S
BJ
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents; D005615:Freezing; D006801:Humans; D007306:Insecticides / Q000633:toxicity; D012967:Sodium Dodecyl Sulfate; D013778:Tetrazolium Salts; D013844:Thiazoles; D014052:Toluidines / Q000633:toxicity; D018675:Toxicity Tests
D004396:Coloring Agents; D007306:Insecticides; D013778:Tetrazolium Salts; D013844:Thiazoles; D014052:Toluidines; C014983:amitraz; D012967:Sodium Dodecyl Sulfate; C022616:thiazolyl blue