title: Identification of live hair cells in rat cochlear sections in culture with FM1-43 fluorescent dye.
abstract: Cochlear hair cells are presumed to live in culture for many days, yet they are difficult to identify in cultured tissues. We stained hair cells in cochlear sections with FM1-43 and cultured them in collagen matrix. Three rows of outer hair cells and a single row of inner ones were distinguished by staining with FM1-43. Fixation of the sections with paraformaldehyde caused loss of the FM1-43 fluorescence, indicating that FM1-43 stained only live hair cells. In sections cultured for 48 h, almost all hair cells were still positive with FM1-43. Culture with gentamycin caused loss of FM1-43-positive cells. In serum-free, long-term cultures (15 days) performed without antibiotics or neurotrophins, the row alignment of FM1-43-positive hair cells was still maintained. Membranous labyrinth-like vacuoles enveloping hair cells were formed in the collagen matrix. Accordingly, FM1-43 is an efficient marker for identifying live hair cells in cultured tissues. Moreover, cochlear hair cells are revealed to live for weeks in serum-free culture without exogenous neurotrophins.
authors.affiliation: Laboratory of Molecular and Cellular Physiology, Department of Physiology, National Defense Medical College of Japan, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. jfukuda@cc.ndmc.ac.jp
authors.forename: Jun
authors.initials: J
authors.lastname: Fukuda
authors.forename: Hisako
authors.initials: H
authors.lastname: Ishimine
authors.forename: Motohide
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers; D018929:Cell Culture Techniques / Q000379:methods*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005109:Extracellular Matrix / Q000502:physiology; D005456:Fluorescent Dyes; D005839:Gentamicins / Q000494:pharmacology; D006198:Hair Cells, Auditory / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008867:Microtomy / Q000379:methods; D009414:Nerve Growth Factors / Q000494:pharmacology; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats
chemical_list: D015415:Biomarkers; D016895:Culture Media, Serum-Free; C073804:FM1 43; D005456:Fluorescent Dyes; D005839:Gentamicins; D009414:Nerve Growth Factors; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds