Institut Curie 9 articles published in JoVE Immunology and Infection Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells Alessandro Donada1, Tamar Tak1, Giulio Prevedello1,2,3,4, Idan Milo1, Ken R. Duffy2, Leïla Perié1 1Quantitative Immuno-hematology, CNRS UMR168, Institut Curie, 2Hamilton Institute, Maynooth University, 3Institut Curie, PSL Research University, CNRS UMR 3348, Orsay, 4Université Paris-Sud, Université Paris-Saclay, CNRS UMR 3348, Orsay Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework. Biology Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C Antonia Hauth1, Rafael Galupa1, Nicolas Servant2, Laura Villacorta1, Kai Hauschulz3, Joke Gerarda van Bemmel4, Agnese Loda1, Edith Heard1,5 1EMBL: European Molecular Biology Laboratory, 2Institut Curie, 3Agilent Technologies, 4Genmab BV, 5Collège de France This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements. Biochemistry Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles Satish Bodakuntla*1,2, A.S. Jijumon*1,2, Carsten Janke1,2, Maria M. Magiera1,2 1PSL Research University, CNRS UMR3348, Institut Curie, 2Université Paris-Saclay, CNRS UMR3348, Université Paris Sud This protocol describes tubulin purification from small/medium-scale sources such as cultured cells or single mouse brains, using polymerization and depolymerization cycles. The purified tubulin is enriched in specific isotypes or has specific posttranslational modifications and can be used in in vitro reconstitution assays to study microtubule dynamics and interactions. Biology Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells Hugo Lachuer1,2,3, Pallavi Mathur1,2,3, Kevin Bleakley4, Kristine Schauer1,2,3 1Unité Mixte de Recherche 144 CNRS, Molecular Mechanisms of Intracellular Transport group, Institut Curie, 75005 Paris, France, 2PSL Research University, Paris, France, 3Sorbonne Université, Paris, France, 4INRIA, Université Paris-Sud, PSL Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes. Developmental Biology Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo Beatrice C. Durand1,2,3,4 1Institut Curie, 2UMR 3387, CNRS, 3PSL Research University, 4Université Paris-Sud In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. Developmental Biology Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models Lee Parry1, Madeleine Young1, Fatima El Marjou2, Alan Richard Clarke1 1European Cancer Stem Cell Research Institute, Cardiff University, 2Institut Curie Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia. Bioengineering Study of Cell Migration in Microfabricated Channels Pablo Vargas1, Emmanuel Terriac2, Ana-Maria Lennon-Duménil1, Matthieu Piel2 1Immunité et Cancer, Institut Curie, 2Compartimentation et Dynamique Cellulaires, Institut Curie A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments. Bioengineering Stretching Micropatterned Cells on a PDMS Membrane Nicolas Carpi1, Matthieu Piel1 1UMR 144, Institut Curie This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching. Medicine Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D Sara Geraldo1, Anthony Simon1, Danijela M. Vignjevic1 1UMR 144 CNRS, Institut Curie This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.
