Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Apoptosis Methods in Toxicology

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

  • 973 Accesses

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

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

References

  1. Hetts SW (1998) To die or not to die: an overview of apoptosis and its role in disease. JAMA 279(4):300–307

    Article  CAS  PubMed  Google Scholar 

  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):358–361

    Article  CAS  PubMed  Google Scholar 

  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):961–967

    CAS  PubMed  Google Scholar 

  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):633–640

    Article  CAS  PubMed  Google Scholar 

  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):4752–4765

    Article  CAS  PubMed  Google Scholar 

  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):654–666

    Article  CAS  PubMed  Google Scholar 

  7. Granato M, Nusslein-Volhard C (1996) Fishing for genes controlling development. Curr Opin Genet Dev 6(4):461–468

    Article  CAS  PubMed  Google Scholar 

  8. National Research Council (2007) Application of toxicogenomic technologies to predictive toxicology and risk assessment. National Academy of Sciences, Washington, DC, p 301

    Google Scholar 

  9. Westerfield M (1993) The zebrafish book: a guide for the laboratory use of zebrafish. The University of Oregon Press, Eugene

    Google Scholar 

  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):243–253

    Article  CAS  PubMed  Google Scholar 

  11. Serbedzija G, Semino C, Frost D et al (2003) Methods of screening agents for activity using teleosts. US Patent 6,656,449

    Google Scholar 

  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 103–116

    Google Scholar 

  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 219–232

    Google Scholar 

  14. Parng C, Ton C, Lin YX et al (2006) A zebrafish assay for identifying neuroprotectants in vivo. Neurotoxicol Teratol 28(4):509–516

    Article  CAS  PubMed  Google Scholar 

  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):7086–7091

    Article  CAS  PubMed  Google Scholar 

  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): 8172–8181

    Article  CAS  PubMed  Google Scholar 

  17. Negron JF, Lockshin RA (2004) Activation of apoptosis and caspase-3 in zebrafish early gastrulae. Dev Dyn 231(1):161–170

    Article  CAS  PubMed  Google Scholar 

  18. Yamashita M (2003) Apoptosis in zebrafish development. Comp Biochem Physiol B Biochem Mol Biol 136(4):731–742

    Article  PubMed  Google Scholar 

  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):e51060

    Google Scholar 

  20. Parng C, Anderson N, Ton C et al (2004) Zebrafish apoptosis assays for drug discovery. Methods Cell Biol 76:75–85

    Article  PubMed  Google Scholar 

  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, MD

    Google Scholar 

  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):67–73

    Article  PubMed  Google Scholar 

  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):151–176

    CAS  PubMed  Google Scholar 

  24. Michael S, Auld D, Klumpp C et al (2008) A robotic platform for quantitative high-throughput screening. Assay Drug Dev Technol 6(5):637–657

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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):24–30

    Article  CAS  PubMed  Google Scholar 

  26. Graham RK, Ehrnhoefer DE, Hayden MR (2011) Caspase-6 and neurodegeneration. Trends Neurosci 34(12):646–656

    Article  CAS  PubMed  Google Scholar 

  27. Jana K, Banerjee B, Parida PK (2013) Caspase: a potential therapeutic targets in the treatment of Alzheimer’s disease. Trans Med S2:006

    Google Scholar 

  28. Gianinazzi C, Grandgirard D, Imboden H et al (2003) Caspase-3 mediates hippocampal apoptosis in pneumococcal meningitis. Acta Neuropathol 105(5):499–507

    CAS  PubMed  Google Scholar 

  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 7:8

    Article  PubMed Central  PubMed  Google Scholar 

  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):1062–1071

    Article  CAS  PubMed  Google Scholar 

  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–H1614

    CAS  PubMed  Google Scholar 

  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):4263–4271

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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):259–266

    Article  CAS  PubMed  Google Scholar 

  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):709–715

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Phylonix, 100 Inman Street, Cambridge, MA, 02139, USA

    Wen Lin Seng, Dawei Zhang & Patricia McGrath

Authors
  1. Wen Lin Seng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dawei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Patricia McGrath
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wen Lin Seng .

Editor information

Editors and Affiliations

  1. Department of Biology, North Carolina A&T State University, Greensboro, North Carolina, USA

    Perpetua M. Muganda

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Seng, W.L., Zhang, D., McGrath, P. (2016). Microplate-Based Whole Zebrafish Caspase 3/7 Assay for Screening Small Molecule Compounds. In: Muganda, P. (eds) Apoptosis Methods in Toxicology. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3588-8_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3588-8_11

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3586-4

  • Online ISBN: 978-1-4939-3588-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation