A fast Resazurin-based live viability assay is equivalent to the MTT-test in the KeratinoSens assay

•Presto-Blue® assay perfectly compatible with luciferase assay in same cell.•Excellent correlation to MTT assay.•Reporter assays such as KeratinoSens ideally use Presto-Blue® assay.•This ensures result is generated on the same cells and reduces variability. 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 R2 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.