Skip to main content
SpringerLink
Log in

High-Throughput Direct Cell Counting Method for Immuno-Oncology Functional Assays Using Image Cytometry

  • Protocol
  • First Online:
Immuno-Oncology

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

Abstract

The last decade has seen significant progress in the field of immuno-oncology. The increase in new technologies has led to the development of novel immuno-oncology assays to investigate immune responses to cancer cells. Numerous cell-based assays have utilized image cytometry due to its abilities to perform high-throughput screening, observe and automatically analyze cells directly in standard microplates, and digitally store the acquired cell images. In this chapter, we describe the use of Celigo Image Cytometer for immuno-oncology functional assays such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), T cell proliferation, and T cell migration.

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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
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

References

  1. Couzin-Frankel J (2013) Cancer immunotherapy. Science 342:1432–1433

    Article  CAS  PubMed  Google Scholar 

  2. Li M, Zheng H, Duan Z, Liu H, Hu D, Bode A, Dong Z, Cao Y (2012) Promotion of cell proliferation and inhibition of ADCC by cancerous immunoglobulin expressed in cancer cell lines. Cell Mol Immunol 9:54–61

    Article  PubMed  Google Scholar 

  3. Somanchi SS, McCulley KJ, Somanchi A, Chan LL, Lee DA (2015) A novel method for assessment of natural killer cell cytotoxicity using image cytometry. PLoS One 10:e0141074

    Article  PubMed  PubMed Central  Google Scholar 

  4. Zaritskaya L, Shurin MR, Sayers TJ, Malyguine AM (2010) New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Rev Vaccines 9:601–616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lindorfer MA, Cook EM, Tupitza JC, Zent CS, Burack R, Jong RNd, Beurskens FJ, Schuurman J, Parren PWHI, Taylor RP (2016) Real-time analysis of the detailed sequence of cellular events in mAb-mediated complement-dependent cytotoxicity of B-cell lines and of chronic lymphocytic leukemia B-cells. Mol Immunol 70:13–23

    Article  CAS  PubMed  Google Scholar 

  6. Riedl T, Boxtel Ev, Bosch M, Parren PWHI, Gerritsen AF (2016) High-throughput screening for internalizing antibodies by homogeneous fluorescence imaging of a pH-activated probe. J Biomol Screen 21:12–23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Fassy J, Tsalkitzi K, Goncalves-Maia M, Braud VM (2017) A real-time cytotoxicity assay as an alternative to the standard chromium-51 release assay for measurement of human NK and T cell cytotoxic activity. Curr Protoc Immunol 118:7.42.1–7.42.12

    Article  CAS  Google Scholar 

  8. Cerignoli F, Abassi YA, Lamarche BJ, Guenther G, Ana DS, Guimet D, Zhang W, Zhang J, Xi B (2018) In vitro immunotherapy potency assays using real-time cell analysis. PLoS One 13:e0193498

    Article  PubMed  PubMed Central  Google Scholar 

  9. David JM, Dominguez C, McCampbell KK, Gulley JL, Schlom J, Palena C (2017) A novel bifunctional anti-PD-L1/TGF-β Trap fusion protein (M7824) efficiently reverts mesenchymalization of human lung cancer cells. OncoImmunology 6:e1349589

    Article  PubMed  PubMed Central  Google Scholar 

  10. Fantini M, David JM, Saric O, Dubeykovskiy A, Cui Y, Mavroukakis SA, Bristol A, Annunziata CM, Tsang KY, Arlen PM (2018) Preclinical characterization of a novel monoclonal antibody NEO-201 for the treatment of human carcinomas. Front Immunol 8:1899

    Article  PubMed  PubMed Central  Google Scholar 

  11. Mazor Y, Sachsenmeier KF, Yang C, Hansen A, Filderman J, Mulgrew K, Wu H, Dall’Acqua WF (2017) Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence. Sci Rep 7:Article ID 40098

    Article  Google Scholar 

  12. Mazor Y, Yang C, Borrok MJ, Ayriss J, Aherne K, Wu H, Dall’Acqua WF (2016) Enhancement of immune effector functions by modulating IgG’s intrinsic affinity for target antigen. PLoS One 11:e0157788

    Article  PubMed  PubMed Central  Google Scholar 

  13. Clay TM, Hobeika AC, Mosca PJ, Lyerly HK, Morse MA (2001) Assays for monitoring cellular immune responses to active immunotherapy of cancer. Clin Cancer Res 7:1127–1135

    CAS  PubMed  Google Scholar 

  14. Sievers EL, Senter PD (2013) Antibody-drug conjugates in cancer therapy. Annu Rev Med 64:15–29

    Article  CAS  PubMed  Google Scholar 

  15. Duensing TD, Watson SR (2018) Complement-dependent cytotoxicity assay. Cold Spring Harb Protoc 2018(2). https://doi.org/10.1101/pdb.prot093799

  16. Tseng D, Volkmer J-P, Willingham SB, Contreras-Trujillo H, Fathman JW, Fernhoff NB, Seita J, Inlay MA, Weiskopf K, Miyanishi M, Weissman IL (2013) Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response. Proc Natl Acad Sci U S A 110:11103–11108. https://doi.org/10.1073/pnas.1305569110

    Article  PubMed  PubMed Central  Google Scholar 

  17. Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart A (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148:3–15

    Article  CAS  PubMed  Google Scholar 

  18. Kunz-Schughart LA, Freyer JP, Hofstaedter F, Ebner R (2004) The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. SLAS Discov 9:273–285

    CAS  Google Scholar 

  19. Thoma CR, Zimmermann M, Agarkova I, Kelm J, Krek W (2014) 3D cell culture systems modeling tumor growth determinants in cancer target discovery. Adv Drug Deliv Rev 69-70:29–41

    Article  CAS  PubMed  Google Scholar 

  20. Yip A, Webster RM (2018) The market for chimeric antigen receptor T cell therapies. Nat Rev Drug Discov 17:161–162

    Article  CAS  PubMed  Google Scholar 

  21. Chan LL-Y, Smith T, Kumph KA, Kuksin D, Kessel S, Déry O, Cribbes S, Lai N, Qiu J (2016) A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry. Cytotechnology 68:2015–2025

    Article  PubMed  PubMed Central  Google Scholar 

  22. Zhang H, Chan LL-Y, Rice W, Kassam N, Longhi MS, Zhao H, Robson SC, Gao W, Wu Y (2017) Novel high-throughput cell-based hybridoma screening methodology using the Celigo Image Cytometer. J Immunol Methods 447:23–30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The author would like to thank the external scientists of Nexcelom Bioscience LLC. for providing immuno-oncology experimental results from the Celigo Image Cytometer.

Conflicts of Interest: The author, L.L.C., declares competing financial interests. The methods and experiments presented in this book chapter were developed and performed using the Celigo Image Cytometer, which is a product of Nexcelom Bioscience, L.L.C. The experiments were performed to demonstrate novel in vitro immunotherapy functional assays that significantly improve upon assays currently performed in the field of immuno-oncology.

Author information

Authors and Affiliations

  1. Nexcelom Bioscience LLC, Lawrence, MA, USA

    Leo Li-Ying Chan

Authors
  1. Leo Li-Ying Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Leo Li-Ying Chan .

Editor information

Editors and Affiliations

  1. Elstar Therapeutics, Cambridge, MA, USA

    Seng-Lai Tan

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Chan, L.LY. (2020). High-Throughput Direct Cell Counting Method for Immuno-Oncology Functional Assays Using Image Cytometry. In: Tan, SL. (eds) Immuno-Oncology. Methods in Pharmacology and Toxicology. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0171-6_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0171-6_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0170-9

  • Online ISBN: 978-1-0716-0171-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation