Case Study on Live Cell Apoptosis-Assay Using Lamin-Chromobody Cell-Lines for High-Content Analysis

  • Kourosh Zolghadr
  • Jacqueline Gregor
  • Heinrich Leonhardt
  • Ulrich RothbauerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 911)


The understanding of cellular processes and their physiopathological alterations requires comprehensive data on the abundance, distribution, modification and interaction of cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not on endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain antibodies (VHH) from Camelidae. We have demonstrated that these VHH domains can be fused with fluorescent proteins and expressed in living cells. Those fluorescent antigen-binding proteins-called chromobodies-can be used to detect and trace proteins and other cellular components in vivo. In principle chromobodies can detect any antigenic structure including posttranslational modifications or nonprotein components and thereby dramatically expand the quality and quantity of information that can be gathered in high-content analyses. Here we demonstrate the suitability of this technology to follow apoptosis in living cells in real time.

Key words

Antibodies Chromobodies High-content analysis GFP Fluorescent proteins Live cell microscopy Apoptosis HCA assay 



The authors declare competing financial interests. This work was supported by the GO-Bio Program of the BMBF (Federal Ministry of Science, Germany).


  1. 1.
    Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516CrossRefGoogle Scholar
  2. 2.
    Neamati N, Fernandez A, Wright S, Kiefer J, McConkey DJ (1995) Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. J Immunol 154:3788–3795Google Scholar
  3. 3.
    Oberhammer FA, Hochegger K, Froschl G, Tiefenbacher R, Pavelka M (1994) Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase. J Cell Biol 126:827–837CrossRefGoogle Scholar
  4. 4.
    Biocca S, Pierandrei-Amaldi P, Cattaneo A (1993) Intracellular expression of anti-p21ras single chain Fv fragments inhibits meiotic maturation of Xenopus oocytes. Biochem Biophys Res Commun 197:422–427CrossRefGoogle Scholar
  5. 5.
    Cardinale A, Lener M, Messina S, Cattaneo A, Biocca S (1998) The mode of action of Y13-259 scFv fragment intracellularly expressed in mammalian cells. FEBS Lett 439:197–202CrossRefGoogle Scholar
  6. 6.
    Marasco WA, Chen S, Richardson JH, Ramstedt U, Jones SD (1998) Intracellular antibodies against HIV-1 envelope protein for AIDS gene therapy. Hum Gene Ther 9:1627–1642CrossRefGoogle Scholar
  7. 7.
    Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448CrossRefGoogle Scholar
  8. 8.
    Rothbauer U, Zolghadr K, Muyldermans S, Schepers A, Cardoso MC, Leonhardt H (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7:282–289Google Scholar
  9. 9.
    Muyldermans S, Baral TN, Retamozzo VC, De Baetselier P, De Genst E, Kinne J, Leonhardt H, Magez S, Nguyen VK, Revets H, Rothbauer U, Stijlemans B, Tillib S, Wernery U, Wyns L, Hassanzadeh-Ghassabeh G, Saerens D (2009) Camelid immunoglobulins and nanobody technology. Vet Immunol Immunopathol 128:178–183CrossRefGoogle Scholar
  10. 10.
    Olichon A, Schweizer D, Muyldermans S, de Marco A (2007) Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains. BMC Biotechnol 7:7CrossRefGoogle Scholar
  11. 11.
    Sheriff S, Chang CY, Jeffrey PD, Bajorath J (1996) X-ray structure of the uncomplexed anti-tumor antibody BR96 and comparison with its antigen-bound form. J Mol Biol 259:938–946CrossRefGoogle Scholar
  12. 12.
    van der Linden RH, Frenken LG, de Geus B, Harmsen MM, Ruuls RC, Stok W, de Ron L, Wilson S, Davis P, Verrips CT (1999) Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim Biophys Acta 1431:37–46CrossRefGoogle Scholar
  13. 13.
    Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, Backmann N, Conrath K, Muyldermans S, Cardoso MC, Leonhardt H (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3:887–889CrossRefGoogle Scholar
  14. 14.
    Susin SA, Daugas E, Ravagnan L, Samejima K, Zamzami N, Loeffler M, Costantini P, Ferri KF, Irinopoulou T, Prevost MC, Brothers G, Mak TW, Penninger J, Earnshaw WC, Kroemer G (2000) Two distinct pathways leading to nuclear apoptosis. J Exp Med 192:571–580CrossRefGoogle Scholar
  15. 15.
    Kihlmark M, Imreh G, Hallberg E (2001) Sequential degradation of proteins from the nuclear envelope during apoptosis. J Cell Sci 114:3643–3653Google Scholar
  16. 16.
    Chae HJ, Kang JS, Byun JO, Han KS, Kim DU, Oh SM, Kim HM, Chae SW, Kim HR (2000) Molecular mechanism of staurosporine-induced apoptosis in osteoblasts. Pharmacol Res 42:373–381CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Kourosh Zolghadr
    • 1
    • 2
  • Jacqueline Gregor
    • 2
  • Heinrich Leonhardt
    • 3
  • Ulrich Rothbauer
    • 1
    • 4
    Email author
  1. 1.Natural and Medical Science Institute at the University of TuebingenUniversity of TuebingenReutlingenGermany
  2. 2.ChromoTek GmbHMartinsriedGermany
  3. 3.CIPS, Center for Integrated Protein Science at the Department of Biology IILudwig Maximilians University MunichPlanegg-MartinsriedGermany
  4. 4.ChromoTek GmbHPlanegg-MartinsriedGermany

Personalised recommendations