569 Dirk Saerens and Serge Muyldermans (eds.), Single Domain Antibodies: Methods and Protocols, Methods in Molecular Biology, vol. 911, DOI 10.1007/978-1-61779-968-6_36, © Springer Science+Business Media, LLC 2012 Chapter 36 Case Study on Live Cell Apoptosis-Assay Using Lamin-Chromobody Cell-Lines for High-Content Analysis Kourosh Zolghadr, Jacqueline Gregor, Heinrich Leonhardt, and Ulrich Rothbauer Abstract 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 endog- enous 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 In the early stage of pharmaceutical drug development, high-content analysis (HCA) is an important technology, aiming at maximal readout of cellular parameters in both, quality and quantity. This way, compound libraries are screened and analyzed for desired effects and/or possible side effects at cellular level. Based on high- throughput microscopy with automated image acquisition and computational pattern recognition in multi-well formats, HCA facilitates the simultaneous investigation of multiple cellular 1. Introduction