Skip to main content

A Triple-Parameter-Based Laboratory-Friendly Fluorescence Imaging to Identify Apoptosis in Live Cells

  • Protocol
  • First Online:
Detection of Cell Death Mechanisms

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2255))

Abstract

Cellular signals to resist apoptosis have been attributed as one of the mechanisms of tumorigenesis. Hence, apoptosis is a cardinal target for drug development in cancers, and several antitumor drugs have been designed to induce apoptosis in tumor cells. Recently, venetoclax, a Bcl2 inhibitor that induces apoptosis, has been approved by the FDA for the treatment of CLL and SLL patients. Proapoptotic antitumor drugs have been traditionally developed and tested, targeting apoptosis in tumor cells. The mechanism of such drug actions has been functionally connected to the mechanism of apoptosis. The identification of apoptosis in a tumor cell takes into account different characteristics in several steps of apoptosis. Thus, it is understandable that modes of identification of apoptosis observed in tumor cells in a laboratory have also been tuned to different characteristics in several parameters of apoptosis. Here, we present a detailed methodology for a triple-parameter-based co-fluorescence imaging to identify apoptosis in live tumor cells. The procedure involves co-fluorescence staining specific for three cardinal features of apoptosis in live cells. The procedure is simple, time-sensitive, and can be performed successfully in a laboratory-friendly manner.

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

Access this chapter

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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

Similar content being viewed by others

References

  1. Tomasetti C, Li L, Vogelstein B (2017) Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science 355(6331):1330–1334. https://doi.org/10.1126/science.aaf9011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Macheret M, Halazonetis TD (2015) DNA replication stress as a hallmark of cancer. Annu Rev Pathol 10:425–448. https://doi.org/10.1146/annurev-pathol-012414-040424

    Article  CAS  PubMed  Google Scholar 

  3. Lockshin RA, Zakeri Z (2007) Cell death in health and disease. J Cell Mol Med 11(6):1214–1224. https://doi.org/10.1111/j.1582-4934.2007.00150.x

    Article  PubMed  PubMed Central  Google Scholar 

  4. Mates JM, Segura JA, Alonso FJ, Marquez J (2012) Oxidative stress in apoptosis and cancer: an update. Arch Toxicol 86(11):1649–1665. https://doi.org/10.1007/s00204-012-0906-3

    Article  CAS  PubMed  Google Scholar 

  5. Fulda S, Vucic D (2012) Targeting IAP proteins for therapeutic intervention in cancer. Nat Rev Drug Discov 11(2):109–124. https://doi.org/10.1038/nrd3627

    Article  CAS  PubMed  Google Scholar 

  6. Silke J, Vucic D (2014) IAP family of cell death and signaling regulators. Methods Enzymol 545:35–65. https://doi.org/10.1016/B978-0-12-801430-1.00002-0

    Article  CAS  PubMed  Google Scholar 

  7. Fulda S (2010) Evasion of apoptosis as a cellular stress response in cancer. Int J Cell Biol 2010:370835. https://doi.org/10.1155/2010/370835

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Herr I, Debatin KM (2001) Cellular stress response and apoptosis in cancer therapy. Blood 98(9):2603–2614. https://doi.org/10.1182/blood.v98.9.2603

    Article  CAS  PubMed  Google Scholar 

  9. Fulda S (2015) Targeting extrinsic apoptosis in cancer: challenges and opportunities. Semin Cell Dev Biol 39:20–25. https://doi.org/10.1016/j.semcdb.2015.01.006

    Article  CAS  PubMed  Google Scholar 

  10. Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D’Orazi G (2016) Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging 8(4):603–619. https://doi.org/10.18632/aging.100934

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hassan M, Watari H, AbuAlmaaty A, Ohba Y, Sakuragi N (2014) Apoptosis and molecular targeting therapy in cancer. Biomed Res Int 2014:150845. https://doi.org/10.1155/2014/150845

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wong RS (2011) Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 30:87. https://doi.org/10.1186/1756-9966-30-87

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Goldar S, Khaniani MS, Derakhshan SM, Baradaran B (2015) Molecular mechanisms of apoptosis and roles in cancer development and treatment. Asian Pac J Cancer Prev 16(6):2129–2144. https://doi.org/10.7314/apjcp.2015.16.6.2129

    Article  PubMed  Google Scholar 

  14. Kaczanowski S (2016) Apoptosis: its origin, history, maintenance and the medical implications for cancer and aging. Phys Biol 13(3):031001. https://doi.org/10.1088/1478-3975/13/3/031001

    Article  CAS  PubMed  Google Scholar 

  15. Sun G, Guzman E, Balasanyan V, Conner CM, Wong K, Zhou HR, Kosik KS, Montell DJ (2017) A molecular signature for anastasis, recovery from the brink of apoptotic cell death. J Cell Biol 216(10):3355–3368. https://doi.org/10.1083/jcb.201706134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tang HL, Yuen KL, Tang HM, Fung MC (2009) Reversibility of apoptosis in cancer cells. Br J Cancer 100(1):118–122. https://doi.org/10.1038/sj.bjc.6604802

    Article  CAS  PubMed  Google Scholar 

  17. Diaz D, Prieto A, Reyes E, Barcenilla H, Monserrat J, Alvarez-Mon M (2015) Flow cytometry enumeration of apoptotic cancer cells by apoptotic rate. Methods Mol Biol 1219:11–20. https://doi.org/10.1007/978-1-4939-1661-0_2

    Article  CAS  PubMed  Google Scholar 

  18. De P, Sun Y, Carlson JH, Friedman LS, Leyland-Jones BR, Dey N (2014) Doubling down on the PI3K-AKT-mTOR pathway enhances the anti-tumor efficacy of PARP inhibitor in triple negative breast cancer model beyond BRCA-ness. Neoplasia 16(1):43–72. https://doi.org/10.1593/neo.131694

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dey N, Sun Y, Carlson JH, Wu H, Lin X, Leyland-Jones B, De P (2016) Anti-tumor efficacy of BEZ235 is complemented by its anti-angiogenic effects via downregulation of PI3K-mTOR-HIF1alpha signaling in HER2-defined breast cancers. Am J Cancer Res 6(4):714–746

    CAS  PubMed  PubMed Central  Google Scholar 

  20. De P, Carlson JH, Leyland-Jones B, Williams C, Dey N (2018) Triple fluorescence staining to evaluate mechanism-based apoptosis following chemotherapeutic and targeted anti-cancer drugs in live tumor cells. Sci Rep 8(1):13192. https://doi.org/10.1038/s41598-018-31575-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Nandini Dey .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

De, P., Aske, J.C., Dey, N. (2021). A Triple-Parameter-Based Laboratory-Friendly Fluorescence Imaging to Identify Apoptosis in Live Cells. In: Alvero, A.B., Mor, G.G. (eds) Detection of Cell Death Mechanisms. Methods in Molecular Biology, vol 2255. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1162-3_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1162-3_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1161-6

  • Online ISBN: 978-1-0716-1162-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics