BCL-2 Family Proteins pp 163-172 | Cite as
Quantification of BCL-2 Family Members by Flow Cytometry
Protocol
First Online:
- 1.1k Downloads
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-XLReferences
- 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.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.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.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.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.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.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.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.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.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.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.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.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.Bordeaux J, Welsh A, Agarwal S, Killiam E, Baquero M, Hanna J et al (2010) Antibody validation. BioTechniques 48:197–209CrossRefPubMedPubMedCentralGoogle Scholar
- 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.Craig FE, Foon KA (2008) Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111:3941–3967CrossRefPubMedGoogle Scholar
- 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.Stewart CC, Stewart SJ (2001) Cell preparation for the identification of leukocytes. Methods Cell Biol 63:217–251CrossRefPubMedGoogle Scholar
- 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