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

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


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 



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


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

Ala (A)



Acridine orange

Asp (D)

Aspartic acid


Aspartic acid–glutamic acid–valine–aspartic acid


American Veterinary Medical Association


Coefficient of variation


Dimethyl sulfoxide


Days post fertilization


European Centre for the Validation of Alternative Methods

Glu (E)

Glutaric acid




Hours post-fertilization


Human umbilical vein endothelial cells




Ethyl 3-aminobenzoate methanesulfonate






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


Relative luminescence units


Region of Interest




Standard deviation



Val (V)







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



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.


  1. 1.
    Hetts SW (1998) To die or not to die: an overview of apoptosis and its role in disease. JAMA 279(4):300–307CrossRefPubMedGoogle Scholar
  2. 2.
    Lamkanfi M, Declercq W, Kalai M et al (2002) Alice in caspase land. A phylogenetic analysis of caspases from worm to man. Cell Death Differ 9(4):358–361CrossRefPubMedGoogle Scholar
  3. 3.
    Uren AG, O’Rourke K, Aravind LA et al (2000) Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol Cell 6(4):961–967PubMedGoogle Scholar
  4. 4.
    Chakraborty C, Nandi SS, Sinha S et al (2006) Zebrafish caspase-3: molecular cloning, characterization, crystallization and phylogenetic analysis. Protein Pept Lett 13(6):633–640CrossRefPubMedGoogle Scholar
  5. 5.
    Agniswamy J, Fang B, Weber IT (2007) Plasticity of S2–S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis. FEBS J 274(18):4752–4765CrossRefPubMedGoogle Scholar
  6. 6.
    Fang B, Boross PI, Tozser J et al (2006) Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition. J Mol Biol 360(3):654–666CrossRefPubMedGoogle Scholar
  7. 7.
    Granato M, Nusslein-Volhard C (1996) Fishing for genes controlling development. Curr Opin Genet Dev 6(4):461–468CrossRefPubMedGoogle Scholar
  8. 8.
    National Research Council (2007) Application of toxicogenomic technologies to predictive toxicology and risk assessment. National Academy of Sciences, Washington, DC, p 301Google Scholar
  9. 9.
    Westerfield M (1993) The zebrafish book: a guide for the laboratory use of zebrafish. The University of Oregon Press, EugeneGoogle Scholar
  10. 10.
    Seng WL, Eng K, Lee J et al (2004) Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment. Angiogenesis 7(3):243–253CrossRefPubMedGoogle Scholar
  11. 11.
    Serbedzija G, Semino C, Frost D et al (2003) Methods of screening agents for activity using teleosts. US Patent 6,656,449Google Scholar
  12. 12.
    Li C, Luo L, Awerman J et al (2012) Whole zebrafish cytochrome P450 assay for assessing drug metabolism and safety. In: McGrath P (ed) Zebrafish, methods for assessing drug safety and toxicity. Wiley, Hoboken, NJ, pp 103–116Google Scholar
  13. 13.
    Li C, Luo L, McGrath P (2012) Zebrafish xenotransplant cancer model for drug screening. In: McGrath P (ed) Zebrafish, methods for assessing drug safety and toxicity. Wiley, Hoboken, NJ, pp 219–232Google Scholar
  14. 14.
    Parng C, Ton C, Lin YX et al (2006) A zebrafish assay for identifying neuroprotectants in vivo. Neurotoxicol Teratol 28(4):509–516CrossRefPubMedGoogle Scholar
  15. 15.
    Daroczi B, Kari G, McAleer MF et al (2006) In vivo radioprotection by the fullerene nanoparticle DF-1 as assessed in a zebrafish model. Clin Cancer Res 12(23):7086–7091CrossRefPubMedGoogle Scholar
  16. 16.
    Geiger GA, Parker SE, Beothy AP et al (2006) Zebrafish as a “biosensor”? Effects of ionizing radiation and amifostine on embryonic viability and development. Cancer Res 66(16): 8172–8181CrossRefPubMedGoogle Scholar
  17. 17.
    Negron JF, Lockshin RA (2004) Activation of apoptosis and caspase-3 in zebrafish early gastrulae. Dev Dyn 231(1):161–170CrossRefPubMedGoogle Scholar
  18. 18.
    Yamashita M (2003) Apoptosis in zebrafish development. Comp Biochem Physiol B Biochem Mol Biol 136(4):731–742CrossRefPubMedGoogle Scholar
  19. 19.
    Sorrells S, Toruno C, Stewart RA et al (2013) Analysis of apoptosis in zebrafish embryos by whole-mount immunofluorescence to detect activated caspase 3. J Vis Exp (82):e51060Google Scholar
  20. 20.
    Parng C, Anderson N, Ton C et al (2004) Zebrafish apoptosis assays for drug discovery. Methods Cell Biol 76:75–85CrossRefPubMedGoogle Scholar
  21. 21.
    Iversen PW, Beck B, Chen Y-F et al (2012) HTS assay validation. In: Sittampalam GS, Gal-Edd N, Arkin MEA (eds) HTS assay validation. Eli Lilly & Company and the National Center for Advancing Translational Services, Bethesda, MDGoogle Scholar
  22. 22.
    Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73CrossRefPubMedGoogle Scholar
  23. 23.
    Genschow E, Spielmann H, Scholz G et al (2002) The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern Lab Anim 30(2):151–176PubMedGoogle Scholar
  24. 24.
    Michael S, Auld D, Klumpp C et al (2008) A robotic platform for quantitative high-throughput screening. Assay Drug Dev Technol 6(5):637–657CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Chang H, Schimmer AD (2007) Livin/melanoma inhibitor of apoptosis protein as a potential therapeutic target for the treatment of malignancy. Mol Cancer Ther 6(1):24–30CrossRefPubMedGoogle Scholar
  26. 26.
    Graham RK, Ehrnhoefer DE, Hayden MR (2011) Caspase-6 and neurodegeneration. Trends Neurosci 34(12):646–656CrossRefPubMedGoogle Scholar
  27. 27.
    Jana K, Banerjee B, Parida PK (2013) Caspase: a potential therapeutic targets in the treatment of Alzheimer’s disease. Trans Med S2:006Google Scholar
  28. 28.
    Gianinazzi C, Grandgirard D, Imboden H et al (2003) Caspase-3 mediates hippocampal apoptosis in pneumococcal meningitis. Acta Neuropathol 105(5):499–507PubMedGoogle Scholar
  29. 29.
    Yoshimori A, Sakai J, Sunaga S et al (2007) Structural and functional definition of the specificity of a novel caspase-3 inhibitor, Ac-DNLD-CHO. BMC Pharmacol 7:8CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Renolleau S, Fau S, Goyenvalle C et al (2007) Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender. J Neurochem 100(4):1062–1071CrossRefPubMedGoogle Scholar
  31. 31.
    Fauvel H, Marchetti P, Chopin C et al (2001) Differential effects of caspase inhibitors on endotoxin-induced myocardial dysfunction and heart apoptosis. Am J Physiol Heart Circ Physiol 280(4):H1608–H1614PubMedGoogle Scholar
  32. 32.
    Voth DE, Howe D, Heinzen RA (2007) Coxiella burnetii inhibits apoptosis in human THP-1 cells and monkey primary alveolar macrophages. Infect Immun 75(9):4263–4271CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Yang Y, Yang L, You QD et al (2007) Differential apoptotic induction of gambogic acid, a novel anticancer natural product, on hepatoma cells and normal hepatocytes. Cancer Lett 256(2):259–266CrossRefPubMedGoogle Scholar
  34. 34.
    Kawamura T, Liu D, Towle MJ et al (2003) Anti-angiogenesis effects of borrelidin are mediated through distinct pathways: threonyl-tRNA synthetase and caspases are independently involved in suppression of proliferation and induction of apoptosis in endothelial cells. J Antibiot (Tokyo) 56(8):709–715CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.PhylonixCambridgeUSA

Personalised recommendations