title: Improving the interferences of methyl thiazolyl tetrazolium and IL-8 assays in assessing the cytotoxicity of nanoparticles.
abstract: Methyl thiazolyl tetrazolium (MTT) and interleukin-8 (IL-8) assays are common colorimetric methods to measure mitochondrial activity and drug induced pro-inflammatory factors. However, many reports have described how MTT absorbance and cytokine adsorption could limit their applicability in evaluating the cytotoxicity of nanomaterials. In this study, we used an acid-containing isopropanol complex as a substitute for dimethyl sulfoxide (DMSO) solvent to dissolve MTT formazan, which was expected to diminish the absorbance of nano-ZnO at 570 nm where maximum absorbance for the MTT formazan was detected. In addition, we used a serum-containing medium to prevent the possible effects of IL-8 protein adsorption in the nano-ZnO and nano-TiO2. The results showed that the modified method by using acid-containing isopropanol step in MTT assay, nano-ZnO exposed to human lung epithelial cells had the lowest cell viability (from 12.5 to 50 microg mL(-1)) and EC50 value (8.4 microg mL(-1)) comparing with the conventional MTT protocol or adding phosphate buffered saline (PBS) to wash cells. The reason for this was the acid-containing isopropanol completely dissolved nano-ZnO with no additional absorbance when compared to the background solvent at 570 nm. On the other hand, the IL-8 protein had a marked influence on the adsorption of nano-TiO2 in the serum-free medium. While only at 100 microg mL(-1) of nano-ZnO, an influence on the adsorption of IL-8 was observed. This could be attributed to the different charges on the surface of nanomaterials. This problem could be overcome through the addition of fetal bovine serum (FBS) to the medium.
authors.affiliation: Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, Taiwan 30013.
authors.forename: I-Lun
authors.initials: IL
authors.lastname: Hsiao
authors.forename: Yuh-Jeen
authors.initials: YJ
authors.lastname: Huang
mesh_terms: D019840:2-Propanol / Q000737:chemistry; D000327:Adsorption / Q000187:drug effects; D001681:Biological Assay; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D006801:Humans; D016209:Interleukin-8 / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity*; D013053:Spectrophotometry; D013778:Tetrazolium Salts / Q000737:chemistry*; D013844:Thiazoles / Q000737:chemistry*; D014025:Titanium / Q000737:chemistry / Q000633:toxicity; D018675:Toxicity Tests; D015034:Zinc Oxide / Q000737:chemistry / Q000633:toxicity
chemical_list: D016209:Interleukin-8; D013778:Tetrazolium Salts; D013844:Thiazoles; C009495:titanium dioxide; D014025:Titanium; C022616:thiazolyl blue; D019840:2-Propanol; D015034:Zinc Oxide; D004121:Dimethyl Sulfoxide