Identification of live hair cells in rat cochlear sections in culture with FM1-43 fluorescent dye.
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.
Fukuda
Jun
J
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
Ishimine
Hisako
H
Tokunaga
Motohide
M
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
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