Microplate-Based Whole Zebrafish Caspase 3/7 Assay for Screening Small Molecule Compounds

In this research, using a commercially available human specific caspase 3/7 chemiluminescent test kit (Caspase 3/7 Glo, Promega, Madison, WI), developed for cell based assays, we describe a microplate-based whole zebrafish assay format to identify potential small molecule caspase inhibitors and activators. Based on the high degree of evolutionary conservation among species, we show that human specific 3/7 substrate cross reacts with zebrafish. Using untreated zebrafish, optimum assay conditions (including substrate concentration, number of zebrafish per microwell, and incubation time to generate a linear reaction) are determined. Robustness and reproducibility of the assay are established using a characterized caspase 3/7 inhibitor (z-VAD-fmk) and an activator (staurosporine). Next, the whole zebrafish microplate assay format is validated using three additional characterized caspase 3/7 inhibitors, two additional caspase 3/7 activators, and one control compound that has no effect on zebrafish apoptosis. Compared to other whole animal assay formats, chemiluminescence provides high sensitivity and low background. Next, results are compared with published results in mammalian cell based assays and animal models and show that the overall predictive success rate is 100 %. Compound effects on apoptosis are further confirmed visually by whole mount staining with acridine orange (AO), a live dye. Results support the high degree of conservation of key pathways in zebrafish and humans....
Source: Springer protocols feed by Pharmacology/Toxicology - Category: Drugs & Pharmacology Source Type: news