As in other vertebrates, apoptosis in zebrafish is a genetically programmed process of cell suicide that occurs in normal physiological processes and in response to apoptosis-inducing signals. We have developed visual and quantitative apoptosis assays using zebrafish as a model system. Whole-animal assays using zebrafish have many advantages, including: short assay time, single dosing, small amount of drug required for each test, statistically significant number of animals for each condition, and low cost.

Visual vital dye assay: The sites and levels of apoptosis can be evaluated in vivo by labeling with vital dyes such as acridine orange. Drug effects in suppressing or inducing apoptosis are detected by the changes in the number of acridine orange-labeled cells (example shown in Figure 1).

Figure 1. Detection of drug-induced apoptosis in vivo using acridine orange. (A), control. (B), zebrafish treated with caffeine at 15 µg/ml. Note the increase of apoptosis in the brain after exposure to high concentration of caffeine (arrow).

Quantitative apoptosis assay in 96-well microplate format: The level of apoptosis is quantitated in a high-throughput manner by extracting the fluorescence dye from the labeled zebrafish, and measuring the fluorescence with a plate reader (examples shown in Figure 2). This quantitative in vivo apoptosis assay is very suitable for screening compound libraries.

Figure 2. Dose-dependent apoptosis can be quantitated by a microplate assay. Zebrafish embryos were treated with neomycin, followed by acridine orange staining and fluorescence measurement. Neomycin induced apoptosis in a dose-dependent manner.