Evaluation of Alamar Blue reduction for the in vitro assay of hepatocyte toxicity

Alamar Blue (AB) reduction is a promising new in vitro assay which is simpl e to conduct and amenable to repeated measurements and high-throughput scre ening: however, evaluation with hepatocytes has not been reported. Accordin gly, we compared AB reduction with established markers of hepatocyte viabil ity and cell density. Primary rat hepatocytes were allowed to adhere to col lagen-coated 96-well plates, then exposed for 16 hours to culture medium, 0 .7% dimethyl sulfoxide (DMSO) in medium, 500 mu M CCl4, 500 mu M eugenol or 15 or 150 mu M of a novel substituted indoline (the latter three in medium with 0.7% DMSO; medium also contained hydrocortisone during exposure perio d). Using a spectrophotometric plate reader. AB reduction was compared with lactate dehydrogenase release (LDH) release, neutral red (NR) uptake, tota l protein (TP) and cell seed density. AB reduction showed a linear relation ship and good correlation with NR uptake, LDH release, TP and cell density. AB assay precision varied with cell density, but was similar to other assa ys in cytotoxicity screening, Good correlation with cell density indicates AB to have the potential for assessment of hepatocyte proliferation. From t he results reported here. we recommend further evaluation and optimization of a protocol for application of AB reduction as a test for cytotoxicity an d proliferation in primary hepatocyte cultures. (C) 1999 Elsevier, Science Ltd. All rights reserved.