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

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Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

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. The microplate-based whole zebrafish caspase 3/7 assay format represents a rapid, reproducible, predictive animal model for identifying potential inhibitors and activators. Use of zebrafish as an alternative animal model to identify potential apoptosis modulators can accelerate the drug discovery process and reduce costs.

Key words

Acridine orange Assay Apoptosis Caspase Chemiluminescence ELISA Methods Microplate Whole zebrafish 

Abbreviations

ac-DEVD-cho

Acetyl-aspartic acid-glutamic acid-valine-aspartic acid-aldehyde

ac-DNLD-cho

Acetyl-aspartic acid-aspartic acid-leucine-aspartic acid-aldehyde

Ala (A)

Alanine

AO

Acridine orange

Asp (D)

Aspartic acid

Asp-Glu-Val-Asp

Aspartic acid–glutamic acid–valine–aspartic acid

AVMA

American Veterinary Medical Association

CV

Coefficient of variation

DMSO

Dimethyl sulfoxide

dpf

Days post fertilization

ECVAM

European Centre for the Validation of Alternative Methods

Glu (E)

Glutaric acid

h

hours

hpf

Hours post-fertilization

HUVEC

Human umbilical vein endothelial cells

M

Mean

MESAB

Ethyl 3-aminobenzoate methanesulfonate

mg

milligram

min

Minutes

q-VD-oph

Quinoline-valine-aspartic acid-oxo-pentanoic acid hydrate

RLU

Relative luminescence units

ROI

Region of Interest

S

Seconds

SD

Standard deviation

S/N

Signal/noise

Val (V)

Valine

μl

Microliter

μM

Micromole

z-VAD-fmk

N-Benzyloxycarbonyl-valine-alanine-aspartic acid (O-Me)-fluoromethylketone

Notes

Acknowledgement

The research was supported in part by National Institutes of Health grants: National Institute of Medical Sciences: 1R43GM087754.

Conflict of Interest

Both Patricia McGrath and Wen Lin Seng are employees and shareholders of Phylonix.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.PhylonixCambridgeUSA

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