Advertisement

Quantification of BCL-2 Family Members by Flow Cytometry

  • Morey L. SmithEmail author
  • Stephen K. Tahir
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

Flow cytometry is a powerful technique for the detection and quantification of cell surface and intracellular proteins. It enables the ability to measure the expression levels of specific proteins in a cell population of interest without the need to physically separate out the cells from within a heterogeneous population by using the appropriate cell-specific markers. It also requires fewer cells than other traditional techniques such as Western blotting. Here we describe a robust and reproducible method to measure the expression levels of the BCL-2 family members, BCL-2, BCL-XL, and MCL-1 by quantitative flow cytometry (QFCM) using validated antibodies.

Key words

Quantitative flow cytometry method Molecules of equivalent soluble fluorochrome BCL-2 MCL-1 BCL-XL 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Meehan RT, Neale LS, Kraus ET, Stuart CA, Smith ML, Cintron NM (1992) Alterations in human mononuclear leucocyte following space flight. Immunology 76:491–497PubMedPubMedCentralGoogle Scholar
  2. 2.
    Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A (2017) Flow cytometry: basic principles and applications. Crit Rev Biotechnol 37:163–176CrossRefPubMedGoogle Scholar
  3. 3.
    Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, et al (2008) ABT-263: A potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res 68:3421–3428CrossRefPubMedGoogle Scholar
  4. 4.
    Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J et al (2013) ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med 19:202–208CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Smith ML, Chyla B, McKeegan E, Tahir SK (2017) Development of a flow cytometric method for quantification of BCL-2 family members in chronic lymphocytic leukemia and correlation with sensitivity to BCL-2 family inhibitors. Cytometry B Clin Cytom 92:331–339CrossRefPubMedGoogle Scholar
  6. 6.
    Konopleva M, Pollyea DA, Potluri J, Chyla B, Hogdal L, Busman T et al (2016) Efficacy and biological correlates of response in a phase II study of venetoclax monotherapy in patients with acute myelogenous leukemia. Cancer Discov 6:1106–1117CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Czabotar PE, Lessene G, Strasser A, Adams JM (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15:49–63CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ashkenazi A, Fairbrother WJ, Leverson JD, Souers AJ (2017) From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors. Nat Rev Drug Discov 16:273–284CrossRefPubMedGoogle Scholar
  9. 9.
    Wong M, Tan N, Zha J, Peale FV, Yue P, Fairbrother WJ et al (2012) Navitoclax (ABT-263) reduces Bcl-xL-mediated chemo-resistance in ovarian cancer models. Mol Cancer Ther 11:1026–1035CrossRefPubMedGoogle Scholar
  10. 10.
    van Stijn A, Kok A, van der Pol MA, Feller N, Roemen GMJM, Westra AH et al (2003) A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia. Leukemia 17:780–786CrossRefPubMedGoogle Scholar
  11. 11.
    Varela M, Ranuncolo SM, Morand A, Lastiri J, De Kier Joffé EB, Puricelli LI et al (2004) EGF-R and PDGF-R, but not bcl-2, overexpression predict overall survival in patients with low-grade astrocytomas. J Surg Oncol 4:34–40CrossRefGoogle Scholar
  12. 12.
    Marcucci G, Stock W, Dai G, Klisovic RB, Liu S, Klisovic MI et al (2005) Phase I study of oblimersen sodium, an antisense to Bcl-2, in untreated older patients with acute myeloid leukemia: pharmacokinetics, pharmacodynamics, and clinical activity. J Clin Oncol 23:3404–3411CrossRefPubMedGoogle Scholar
  13. 13.
    Ding M, Kaspersson K, Murray D, Bardelle C (2017) High-throughput flow cytometry for drug discovery: principles, applications, and case studies. Drug Discov Today 22:1844–1850CrossRefPubMedGoogle Scholar
  14. 14.
    Bordeaux J, Welsh A, Agarwal S, Killiam E, Baquero M, Hanna J et al (2010) Antibody validation. BioTechniques 48:197–209CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    O'Hara DM, Xu Y, Liang Z, Reddy MP, Wu DY, Litwin V (2011) Recommendations for the validation of flow cytometric testing during drug development: II assays. J Immunol Methods 363:120–134CrossRefPubMedGoogle Scholar
  16. 16.
    Craig FE, Foon KA (2008) Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111:3941–3967CrossRefPubMedGoogle Scholar
  17. 17.
    Rezaei A, Adib M, Mokarian F, Tebianian M, Nassiri R (2003) Leukemia markers expression of peripheral blood vs bone marrow blasts using flow cytometry. Med Sci Monit 9:359–362Google Scholar
  18. 18.
    Stewart CC, Stewart SJ (2001) Cell preparation for the identification of leukocytes. Methods Cell Biol 63:217–251CrossRefPubMedGoogle Scholar
  19. 19.
    Phillips DC, Garrison SP, Jeffers JR, Zambetti GP (2009) Assays to measure p53-dependent and independent apoptosis. Methods Mol Biol 559:143–159CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Research and Development, Oncology DiscoveryAbbVie, Inc.North ChicagoUSA

Personalised recommendations

Cite protocol

Buy options