title: A fast Resazurin-based live viability assay is equivalent to the MTT-test in the KeratinoSens assay.
abstract: The KeratinoSens™ assay was the first cell-based in vitro test in the skin sensitisation adverse outcome pathway to be endorsed by an ECVAM statement. It includes a cell viability assessment, which serves two purposes: It forms part of the prediction model to exclude false-positive irritants and cytotoxicity provides some information on sensitizer potency of chemicals, which can feed into a multivariate potency model. In the KeratinoSens™ protocol, Nrf2-dependent luciferase induction and the MTT-viability assay are performed in parallel plates. Resazurin-based viability assays do not require cell lysis and are compatible with luciferase measurements in the same cells. Here, we performed detailed comparison of the tetrazolium-based MTT assay and the PrestoBlue® assay on 35 reference chemicals tested in the full KeratinoSens™ protocol. Log-transformed IC50 and IC30 values measured with both methods correlate with an R(2) of 0.97 and 0.95. A single chemical showed divergent results and analysis by four different viability assays indicated the PrestoBlue® read-out to be correct. The new more rapid and resource efficient approach has clear advantages: Dose-response curves show lower variability and the two endpoints are measured on the same cells. This approach is a valid addition to or replacement of the MTT-readout in the KeratinoSens™ assay and it is recommended as a general tool for luciferase-based reporter assays.
authors: Givaudan Schweiz AG, Ueberlandstrasse 138, CH-8600 Duebendorf, Switzerland.
authors: Roger
authors: R
authors: Emter
authors: Givaudan Schweiz AG, Ueberlandstrasse 138, CH-8600 Duebendorf, Switzerland. Electronic address: andreas.natsch@givaudan.com.
authors: Andreas
authors: A
authors: Natsch
mesh_terms: D000826:Animal Testing Alternatives; D002470:Cell Survival / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005189:False Positive Reactions; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D007202:Indicators and Reagents; D007509:Irritants / Q000633:toxicity*; D010078:Oxazines; D012871:Skin Diseases / Q000139:chemically induced*; D013778:Tetrazolium Salts; D013844:Thiazoles; D018675:Toxicity Tests / Q000379:methods*; D014966:Xanthenes
chemical_list: D007202:Indicators and Reagents; D007509:Irritants; D010078:Oxazines; D013778:Tetrazolium Salts; D013844:Thiazoles; D014966:Xanthenes; C005843:resazurin; C022616:thiazolyl blue
keywords: KeratinoSens™; Luciferase; MTT; PrestoBlue®; Reporter gene assay