Flow Cytometry-Based Detection and Analysis of BCL-2 Family Proteins and Mitochondrial Outer Membrane Permeabilization (MOMP)

  • Lindsey M. Ludwig
  • Katrina L. Maxcy
  • James L. LaBelleEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)


The BCL-2 family of proteins orchestrates a complex signaling network that governs the balance between cellular survival and death. A comprehensive understanding of the mechanistic interactions between these proteins continues to evolve in normal and malignant cells. The functional variation by individual BCL-2 proteins in different cell types has driven clinical therapeutic development in targeting individual BCL-2 members with the goal of fine-tuning cell death in diseased cells. Given the importance of understanding and validating the effect of activating or inhibiting BCL-2 protein interactions in individual cells, the methods used to measure apoptotic cell death have undergone increased scrutiny. Here, we describe two in vitro flow cytometry-based methods that are useful in measuring BCL-2 proteins and mitochondrial-based cell death in complex cell populations.

Key words

BCL-2 proteins Intracellular staining Flow cytometry Mitochondria outer membrane permeabilization (MOMP) Apoptosis 



We would like to thank Eric E. Smith for graphics in Fig. 2 and 5.


  1. 1.
    Cotter TG (2009) Apoptosis and cancer: the genesis of a research field. Nat Rev Cancer 9(7):501–507CrossRefPubMedGoogle Scholar
  2. 2.
    Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26(4):239–257CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    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(1):49–63CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ludwig LM, Nassin ML, Hadji A, LaBelle JL (2016) Killing two cells with one stone: pharmacologic BCL-2 family targeting for cancer cell death and immune modulation. Front Pediatr 4:135CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, Seto Y, Nagata S (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66(2):233–243CrossRefPubMedGoogle Scholar
  6. 6.
    Danial NN, Korsmeyer SJ (2004) Cell death: critical control points. Cell 116(2):205–219CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Fukuhara S, Rowley JD (1978) Chromosome 14 translocations in non-Burkitt lymphomas. Int J Cancer 22(1):14–21CrossRefPubMedGoogle Scholar
  8. 8.
    Tsujimoto Y, Yunis J, Onorato-Showe L, Erikson J, Nowell PC, Croce CM (1984) Molecular cloning of the chromosomal breakpoint of B-cell lymphomas and leukemias with the t(11;14) chromosome translocation. Science 224(4656):1403–1406CrossRefPubMedGoogle Scholar
  9. 9.
    Vaux DL, Cory S, Adams JM (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335(6189):440–442CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Delbridge AR, Grabow S, Strasser A, Vaux DL (2016) Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies. Nat Rev Cancer 16(2):99–109CrossRefPubMedGoogle Scholar
  11. 11.
    Kim H, Tu HC, Ren D, Takeuchi O, Jeffers JR, Zambetti GP, Hsieh JJ, Cheng EH (2009) Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis. Mol Cell 36(3):487–499CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Happo L, Strasser A, Cory S (2012) BH3-only proteins in apoptosis at a glance. J Cell Sci 125(Pt 5):1081–1087CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Taylor RC, Cullen SP, Martin SJ (2008) Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 9(3):231–241CrossRefPubMedGoogle Scholar
  14. 14.
    Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Castedo M, Cidlowski JA, Ciechanover A, Cohen GM, De Laurenzi V, De Maria R, Deshmukh M, Dynlacht BD, El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P, Gougeon ML, Green DR, Gronemeyer H, Hajnoczky G, Hardwick JM, Hengartner MO, Ichijo H, Jaattela M, Kepp O, Kimchi A, Klionsky DJ, Knight RA, Kornbluth S, Kumar S, Levine B, Lipton SA, Lugli E, Madeo F, Malomi W, Marine JC, Martin SJ, Medema JP, Mehlen P, Melino G, Moll UM, Morselli E, Nagata S, Nicholson DW, Nicotera P, Nunez G, Oren M, Penninger J, Pervaiz S, Peter ME, Piacentini M, Prehn JH, Puthalakath H, Rabinovich GA, Rizzuto R, Rodrigues CM, Rubinsztein DC, Rudel T, Scorrano L, Simon HU, Steller H, Tschopp J, Tsujimoto Y, Vandenabeele P, Vitale I, Vousden KH, Youle RJ, Yuan J, Zhivotovsky B, Kroemer G (2009) Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ 16(8):1093–1107CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G (2011) Cell death assays for drug discovery. Nat Rev Drug Discov 10(3):221–237CrossRefPubMedGoogle Scholar
  16. 16.
    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(4):273–284CrossRefPubMedGoogle Scholar
  17. 17.
    Opferman JT (2016) Attacking cancer's Achilles heel: antagonism of anti-apoptotic BCL-2 family members. FEBS J 283(14):2661–2675CrossRefPubMedGoogle Scholar
  18. 18.
    Elmore SP, Nishimura Y, Qian T, Herman B, Lemasters JJ (2004) Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer. Arch Biochem Biophys 422(2):145–152CrossRefPubMedGoogle Scholar
  19. 19.
    Gottlieb E, Armour SM, Harris MH, Thompson CB (2003) Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Differ 10(6):709–717CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Dewson G, Kluck RM (2009) Mechanisms by which Bak and Bax permeabilise mitochondria during apoptosis. J Cell Sci 122(Pt 16):2801–2808CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292(5517):727–730CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Rego AC, Vesce S, Nicholls DG (2001) The mechanism of mitochondrial membrane potential retention following release of cytochrome c in apoptotic GT1-7 neural cells. Cell Death Differ 8(10):995–1003CrossRefPubMedGoogle Scholar
  23. 23.
    Whelan RS, Konstantinidis K, Wei AC, Chen Y, Reyna DE, Jha S, Yang Y, Calvert JW, Lindsten T, Thompson CB, Crow MT, Gavathiotis E, Dorn GW 2nd, O'Rourke B, Kitsis RN (2012) Bax regulates primary necrosis through mitochondrial dynamics. Proc Natl Acad Sci U S A 109(17):6566–6571CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Chen LB (1988) Mitochondrial membrane potential in living cells. Annu Rev Cell Biol 4:155–181CrossRefPubMedGoogle Scholar
  25. 25.
    Cottet-Rousselle C, Ronot X, Leverve X, Mayol JF (2011) Cytometric assessment of mitochondria using fluorescent probes. Cytometry A 79(6):405–425CrossRefPubMedGoogle Scholar
  26. 26.
    Reers M, Smiley ST, Mottola-Hartshorn C, Chen A, Lin M, Chen LB (1995) Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol 260:406–417CrossRefPubMedGoogle Scholar
  27. 27.
    Ryan J, Letai A (2013) BH3 profiling in whole cells by fluorimeter or FACS. Methods 61(2):156–164CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Engbers-Buijtenhuijs P, Kamphuis M, van der Sluijs VG, Haanen C, Poot AA, Feijen J, Vermes I (2005) A novel time resolved fluorometric assay of anoikis using europium-labelled Annexin V in cultured adherent cells. Apoptosis 10(2):429–437CrossRefPubMedGoogle Scholar
  29. 29.
    Paoli P, Giannoni E, Chiarugi P (2013) Anoikis molecular pathways and its role in cancer progression. Biochim Biophys Acta 1833(12):3481–3498CrossRefPubMedGoogle Scholar
  30. 30.
    Bird GH, Gavathiotis E, LaBelle JL, Katz SG, Walensky LD (2014) Distinct BimBH3 (BimSAHB) stapled peptides for structural and cellular studies. ACS Chem Biol 9(3):831–837CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Edwards AL, Wachter F, Lammert M, Huhn AJ, Luccarelli J, Bird GH, Walensky LD (2015) Cellular uptake and Ultrastructural localization underlie the pro-apoptotic activity of a hydrocarbon-stapled BIM BH3 peptide. ACS Chem Biol 10(9):2149–2157CrossRefPubMedGoogle Scholar
  32. 32.
    LaBelle JL, Katz SG, Bird GH, Gavathiotis E, Stewart ML, Lawrence C, Fisher JK, Godes M, Pitter K, Kung AL, Walensky LD (2012) A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers. J Clin Invest 122(6):2018–2031CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Reynolds C, Roderick JE, LaBelle JL, Bird G, Mathieu R, Bodaar K, Colon D, Pyati U, Stevenson KE, Qi J, Harris M, Silverman LB, Sallan SE, Bradner JE, Neuberg DS, Look AT, Walensky LD, Kelliher MA, Gutierrez A (2014) Repression of BIM mediates survival signaling by MYC and AKT in high-risk T-cell acute lymphoblastic leukemia. Leukemia 28(9):1819–1827CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Certo M, Del Gaizo MV, Nishino M, Wei G, Korsmeyer S, Armstrong SA, Letai A (2006) Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 9(5):351–365CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Lindsey M. Ludwig
    • 1
  • Katrina L. Maxcy
    • 1
  • James L. LaBelle
    • 1
    Email author
  1. 1.Section of Hematology/Oncology/Stem Cell Transplantation and Committee on Cancer Biology, Department of PediatricsUniversity of ChicagoChicagoUSA

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