Purification and Immunostaining of Mouse Ependymal Ciliary Shafts
Kai Hao,
Xueliang Zhu ,
Xiumin Yan
Affiliations
Kai Hao
State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, ChinaUniversity of Chinese Academy of Sciences, Beijing 100049, China
Xueliang Zhu
State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, ChinaUniversity of Chinese Academy of Sciences, Beijing 100049, China, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China
Xiumin Yan
Ministry of Education-Shanghai Key Laboratory of Children’s Environmental Health, Institute of Early Life Health, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
Cilia and flagella are microtubule-based hair-like organelles protruding from the surface of most eukaryotic cells, and play essential roles in cell locomotion, left-right asymmetry, embryo development, and tissue homeostasis. With isolated cilia and flagella, great progress has been made in understanding the composition, structure, and function of cilia. However, the current cilia/flagella isolation methods are deficient in the integrity or productivity of purified cilia when applied to mammalian motile cilia. Here, we describe a new protocol that isolates cilia shafts from mouse ependymal cells, by horizontal shear force and mild detergent. This method enables the production of virtually integral cilia with high yields and less cell body contamination. It is suitable for immunostaining, puromycin labeling assay, and proximity ligation assay of mammalian motile cilia.Graphical abstract:
