Induction 96-well plate format

In regards to culturing hepatocytes in a 96-well plate format, we have adopted the same conditions that we used when culturing cells in 60-mm dishes and 24-well plates (12) and simply scaled them down to a 96-well plate format. The 96-well plates are precoated with Matrigel and are commercially available (Collaborative Biomedical Products, Boston, Massachusetts, U.S.) or, alternatively, normal plates can be coated with diluted Matrigel and dried overnight (27). Hepatocytes cultured on collagen-coated 96-well plates have also been reported to be suitable for CYP induction (28). 

We have characterized the activities of several recombinant human and mouse Bcl-2 family proteins (Bcl-2, Bcl-xL, Bcl-w, Bax, Bid, and caspase-8 cleaved Bid) with the cytochrome c ELISA. Dose responses, kinetics, inhibition, and the effects of protein-protein interactions have been quantified. Conducting the cytochrome c release assay in the well of the ELISA plate decreases the time required to perform the assay from 3 h to 35 min. Similar dose responses for induction of cytochrome c release caused by cleaved Bid were obtained with the 35 min and 3 h release assays, indicating that the short format can detect intermediate levels of cytochrome c release. The short format is applicable to automation for screening million-member libraries. Mitochondria obtained from one mouse liver is sufficient to perform 4000 assays in the 96-well plates and scale-up is performed simply by increasing the amount of starting tissue. 

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