CHAPTER SIX Probing Ciliogenesis Using Micropatterned Substrates Amandine Pitaval * ,† , Andreas Christ * , Alexis Curtet * , Qingzong Tseng * , Manuel Théry * ,1 * Laboratoire de Physiologie Cellulaire et Ve ´ge ´tale, Institut de Recherche en Technologies et Sciences pour le Vivant, CNRS/UJF/INRA/CEA, Grenoble, France † Laboratoire Biologie a ` Grande Echelle, Institut de Recherche en Technologies et Sciences pour le Vivant, UJF/INSERM/CEA, Grenoble, France 1 Corresponding author: e-mail address: manuel.thery@cea.fr Contents 1. Introduction 110 2. Micropatterned Substrates Fabrication 112 2.1 Designing features of micropatterns 114 2.2 Micropatterned slides manufacturing 116 3. Cells Plating on Micropatterned Slides 119 3.1 Cell culture 119 3.2 Cell synchronization (facultative) 119 3.3 Cell plating and serum starvation 121 4. Cell Fixation and Immunofluorescence 122 4.1 Cell fixation 123 4.2 Immunostaining 124 5. Automated Image Acquisition 124 6. Automated Image Analysis 125 6.1 Organelle detection on images 126 Acknowledgments 129 References 129 Abstract The primary cilium is a biomechanical sensor plugged in at the cell surface. It is impli- cated in the processing of extracellular signals and its absence or misfunctioning lead to a broad variety of serious defects known as ciliopathies. Unfortunately, the precise mechanisms underlying primary cilium assembly and operation are still poorly under- stood. Molecular dynamics and intracellular morphogenesis are easier to study in cell culture than in tissues. However, cultured cells are usually nonciliated and the empirical methods that are used to induce ciliogenesis in these cells have variable efficiencies. In addition, these methods require cells to be cultured at high density, which is not con- venient for further automated image analysis. Here, we describe a method to induce and modulate ciliogenesis in mammalian cells in culture that is compatible with Methods in Enzymology, Volume 525 # 2013 Elsevier Inc. ISSN 0076-6879 All rights reserved. http://dx.doi.org/10.1016/B978-0-12-397944-5.00006-7 109 Author's personal copy