Sorting of micronuclei from PtK1 cells |
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| Authors: | D Hernandez-Verdun M Grégoire B Labidi M Bouteille |
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| Affiliation: | 1. Laboratoire de Pathologie Cellulaire, 75270 Paris, Cedex 06, France;2. Faculté de Médecine Broussais-Hôtel-Dieu, 75270 Paris, Cedex 06, France;1. Neural Stem Cell Institute, Rensselaer, NY 12144, USA;2. Department of Genetics, University College London Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK;3. NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, City Road, London EC1V 2PD, UK;4. Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA;5. Byers Eye Institute at Stanford University, 2452 Watson Court, Palo Alto, CA 94303, USA;6. Centre for Regenerative Medicine, University of Modena and Reggio Emilia, via G.Gottardi 100, 41125 Modena, Italy;7. Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA;8. The Schepens Eye Research Institute, Massachusetts Eye and Ear, an affiliate of Harvard Medical School, Boston, MA 02114, USA;9. Shiley Eye Institute and Institute for Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA;10. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University and Guangzhou Regenerative Medicine and Health Laboratory, Guangzhou 510060, China;11. Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA;1. FH Aachen UAS, Hohenstaufenallee 6, 52064 Aachen, Germany;2. RMIT University, GPO Box 2476 Melbourne, 3001 Victoria, Australia;1. Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, 3rd Floor, Teaching Research and Wellness Building, 3280 Hospital Drive N.W, Calgary, AB, T2N 4Z6, Canada;2. School of Health Administration, Faculty of Health, Dalhousie University, 5850 College Street, Tupper Building, 2nd Floor, PO Box 15000, Halifax, NS, B3H 4R2, Canada |
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| Abstract: | We report here a procedure allowing to select micronuclei corresponding to defined individualized chromosomes in conditions which preserve their synthetic activity. The mammalian PtK1 cells, which possess six chromosome pairs, were micronucleated by colchicine. DNA of the micronucleated cells was labeled by the Hoechst 33342 fluorochrome under vital conditions. The micronuclei were isolated by a gentle procedure and their fluorescence was analysed by flow cytometry. The flow-cytometry parameters were determined for the analysis of non-fixed subdiploid fractions. We obtained five distinct peaks of fluorescence which have been sorted. The sorted micronuclei are different in each peak exhibiting different fluorescence intensity. Peak 3 contains the micronuclei with nucleoli and chromocenters that correspond to the X chromosome in this cell line. |
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