Analytical and Bioanalytical Chemistry

, Volume 406, Issue 22, pp 5389–5394 | Cite as

A miniaturized device for bioluminescence analysis of caspase-3/7 activity in a single apoptotic cell

  • Eva Adamová
  • Marcela Lišková
  • Eva Matalová
  • Karel KlepárníkEmail author
Research Paper


Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10−15 g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo™ 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 μl of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research.


Bioluminescence detection assembly


Apoptosis Bioluminescence Caspase 3/7 Single-cell analysis 





Tetrapeptide sequence Asp-Glu-Val-Asp


Photomultiplier tube



The research was supported by the Grant Agency of the Czech Republic, projects 14-28254s (at the IAC ASCR, v.v.i.), P304/11/1418 (at the IAPG ASCR, v.v.i.), and P502/12/1285 (at the UVPS). The research runs also under the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101). The authors wish to thank Mgr. Jan Křivánek for the kind gift of human stem cells.


  1. 1.
    Nicholson DW (1999) Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 6:1028–1042CrossRefGoogle Scholar
  2. 2.
    Degterev A, Boyce M, Yuan JY (2003) A decade of caspases. Oncogene 22:8543–8567CrossRefGoogle Scholar
  3. 3.
    Szymczyk KH, Freeman TA, Adams CS, Srinivas V, Steinbeck MJ (2006) Active caspase-3 is required for osteoclast differentiation. J Cell Physiol 209:836–844CrossRefGoogle Scholar
  4. 4.
    Matalova E, Lesot H, Svandova E, Vanden Berghe T, Sharpe PT, Healy C, Vandenabeele P, Tucker AS (2013) Caspase-7 participates in differentiation of cells forming dental hard tissues. Develop Growth Differ 55:615–621CrossRefGoogle Scholar
  5. 5.
    Fujita J, Crane AM, Souza MK, Dejosez M, Kyba M, Flavell RA, Thomson JA, Zwaka TP (2008) Caspase activity mediates the differentiation of embryonic stem cells. Cell Stem Cell 2:595–601CrossRefGoogle Scholar
  6. 6.
    Fernando P, Brunette S, Megeney LA (2005) Neural stem cell differentiation is dependent upon endogenous caspase-3 activity. FASEB J 19:1671–1673Google Scholar
  7. 7.
    Nicholson DW (1996) ICE/CED3-like proteases as therapeutic targets for the control of inappropriate apoptosis. Nat Biotechnol 14:297–301CrossRefGoogle Scholar
  8. 8.
    Thompson CB (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267:1456–1462CrossRefGoogle Scholar
  9. 9.
    Fesik SW (2005) Promoting apoptosis as a strategy for cancer drug discovery. Nat Rev Cancer 5:876–885CrossRefGoogle Scholar
  10. 10.
    Kottke TJ, Blajeski AL, Meng XW, Svingen PA, Ruchaud S, Mesner PW, Boerner SA, Samejima K, Henriquez NV, Chilcote TJ, Lord J, Salmon M, Earnshaw WC, Kaufmann SH (2002) Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells. J Biol Chem 277:804–815CrossRefGoogle Scholar
  11. 11.
    Parton M, Krajewski S, Smith I, Krajewska M, Archer C, Naito M, Ahern R, Reed J, Dowsett M (2002) Coordinate expression of apoptosis-associated proteins in human breast cancer before and during chemotherapy. Clin Cancer Res 8:2100–2108Google Scholar
  12. 12.
    Budihardjo I, Oliver H, Lutter M, Luo X, Wang XD (1999) Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15:269–290CrossRefGoogle Scholar
  13. 13.
    Gown AM, Willingham MC (2002) Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3. J Histochem Cytochem 50:449–454CrossRefGoogle Scholar
  14. 14.
    Duan WR, Garner DS, Williams SD, Funckes-Shippy CL, Spath IS, Blomme EAG (2003) Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J Pathol 199:221–228CrossRefGoogle Scholar
  15. 15.
    Hwang SY, Cho SH, Cho DY, Lee M, Choo J, Jung KH, Maeng JH, Chai YG, Yoon WJ, Lee EK (2011) Time-lapse, single cell based confocal imaging analysis of caspase activation and phosphatidylserine flipping during cellular apoptosis. Biotechnic Histochem 86:181–187CrossRefGoogle Scholar
  16. 16.
    Saito K, Wada I, Tamura M, Kinjo M (2004) Direct detection of caspase-3 activation in single live cells by cross-correlation analysis. Biochem Biophys Res Commun 324:849–854CrossRefGoogle Scholar
  17. 17.
    Pan WL, Qu JL, Chen TS, Sun L, Qi J (2009) FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death. Eur Biophys J Biophys Lett 38:447–456CrossRefGoogle Scholar
  18. 18.
    Gurtu V, Kain SR, Zhang GH (1997) Fluorometric and colorimetric detection of caspase activity associated with apoptosis. Anal Biochem 251:98–102CrossRefGoogle Scholar
  19. 19.
    Saunders PA, Cooper JA, Roodell MM, Schroeder DA, Borchert CJ, Isaacson AL, Schendel MJ, Godfrey KG, Cahill DR, Walz AM, Loegering RT, Gaylord H, Woyno IJ, Kaluyzhny AE, Krzyzek RA, Mortari F, Tsang M, Roff CF (2000) Quantification of active caspase 3 in apoptotic cells. Anal Biochem 284:114–124CrossRefGoogle Scholar
  20. 20.
    Rosell A, Cuadrado E, Alvarez-Sabin J, Hernandez-Guillamon M, Delgado P, Penalba A, Mendioroz M, Rovira A, Fernandez-Cadenas I, Ribo M, Molina CA, Montaner J (2008) Caspase-3 is related to infarct growth after human ischemic stroke. Neurosci Lett 430:1–6CrossRefGoogle Scholar
  21. 21.
    Torkzadeh-Mahani M, Ataei F, Nikkhah M, Hosseinkhani S (2012) Design and development of a whole-cell luminescent biosensor for detection of early-stage of apoptosis. Biosens Bioelectron 38:362–368CrossRefGoogle Scholar
  22. 22.
    Liskova M, Kleparnik K, Matalova E, Hegrova J, Prikryl J, Svandova E, Foret F (2013) Bioluminescence determination of active caspase-3 in single apoptotic cells. Electrophoresis 34:1772–1777CrossRefGoogle Scholar
  23. 23.
    Cheraghi R, Hosseinkhani S, Davoodi J, Nazari M, Amini-Bayat Z, Karimi H, Shamseddin M, Gheidari F (2013) Structural and functional effects of circular permutation on firefly luciferase: in vitro assay of caspase 3/7. Int J Biol Macromol 58:336–342CrossRefGoogle Scholar
  24. 24.
    O’Brien MA, Daily WJ, Hesselberth PE, Moravec RA, Scurria MA, Klaubert DH, Bulleit RF, Wood KV (2005) Homogeneous, bioluminescent protease assays: caspase-3 as a model. J Biomol Screen 10:137–148CrossRefGoogle Scholar
  25. 25.
    Van de Bittner GC, Bertozzi CR, Chang CJ (2013) Strategy for dual-analyte luciferin imaging: in vivo bioluminescence detection of hydrogen peroxide and caspase activity in a murine model of acute inflammation. J Am Chem Soc 135:1783–1795CrossRefGoogle Scholar
  26. 26.
    Niu G, Zhu L, Ho DN, Zhang F, Gao HK, Quan QM, Hida N, Ozawa T, Liu G, Chen XY (2013) Longitudinal bioluminescence imaging of the dynamics of doxorubicin induced apoptosis. Theranostics 3:190–200CrossRefGoogle Scholar
  27. 27.
    Fu QX, Duan XG, Yan SD, Wang LC, Zhou Y, Jia SZ, Du J, Wang XH, Zhang YH, Zhan LS (2013) Bioluminescence imaging of caspase-3 activity in mouse liver. Apoptosis 18:998–1007CrossRefGoogle Scholar
  28. 28.
    Hochgrafe K, Mandelkow EM (2012) Making the brain glow: in vivo bioluminescence imaging to study neurodegeneration. Mol Neurobiol 47:868–882CrossRefGoogle Scholar
  29. 29.
    Hickson J, Ackler S, Klaubert D, Bouska J, Ellis P, Foster K, Oleksijew A, Rodriguez L, Schlessinger S, Wang B, Frost D (2010) Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z-DEVD-aminoluciferin. Cell Death Differ 17:1003–1010CrossRefGoogle Scholar
  30. 30.
    Roth C, Kasimir-Bauer S, Pantel K, Schwarzenbach H (2011) Screening for circulating nucleic acids and caspase activity in the peripheral blood as potential diagnostic tools in lung cancer. Mol Oncol 5:281–291CrossRefGoogle Scholar
  31. 31.
    Chlastakova I, Liskova M, Kudelova J, Dubska L, Kleparnik K, Matalova E (2012) Dynamics of caspase-3 activation and inhibition in embryonic micromasses evaluated by a photon-counting chemiluminescence approach. In Vitro Cell Dev Biol Anim 48:545–549CrossRefGoogle Scholar
  32. 32.
    McStay GP, Salvesen GS, Green DR (2008) Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ 15:322–331CrossRefGoogle Scholar
  33. 33.
    Wong CE, Paratore C, Dours-Zimmermann MT, Rochat A, Pietri T, Suter U, Zimmermann DR, Dufour S, Thiery JP, Meijer D, Beermann F, Barrandon Y, Sommer L (2006) Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J Cell Biol 175:1005–1015CrossRefGoogle Scholar
  34. 34.
    Tucker A, Sharpe P (2004) The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet 5:499–508CrossRefGoogle Scholar
  35. 35.
    Harada H, Kettunen P, Jung HS, Mustonen T, Wang YA, Thesleff I (1999) Localization of putative stem cells in dental epithelium and their association with notch and FGF signaling. J Cell Biol 147:105–120CrossRefGoogle Scholar
  36. 36.
    Musch T, Oz Y, Lyko F, Breiling A (2010) Nucleoside drugs induce cellular differentiation by caspase-dependent degradation of stem cell factors. PLoS One 5, E10726CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Eva Adamová
    • 1
    • 2
    • 3
  • Marcela Lišková
    • 1
  • Eva Matalová
    • 2
    • 3
    • 4
  • Karel Klepárník
    • 1
    Email author
  1. 1.Institute of Analytical Chemistry, v.v.i.Czech Academy of SciencesBrnoCzech Republic
  2. 2.Department of PhysiologyUniversity of Veterinary and Pharmaceutical SciencesBrnoCzech Republic
  3. 3.Institute of Animal Physiology and Genetics, v.v.i.Czech Academy of SciencesBrnoCzech Republic
  4. 4.RECAMOMasaryk Memorial Research InstituteBrnoCzech Republic

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