The cochlear explants were dissected and cultured as previously reported [83 (link),84 (link)]. P3 GFP-LC3B mice were sacrificed by cervical dislocation and soaked in 75% alcohol, and the inner ear tissues including the cochlea were dissected using scissors and placed in pre-cooled sterile HBSS. The volute was opened under a microscope, and the spiral ganglion and vasculature were removed. The cochlea was then attached to the coverslip, which was pretreated with RatCol (Advanced BioMatrix, 5153), and then placed in a four-well dish. Finally, culture media was added and the explants were cultured in an incubator overnight. LPS (Sigma-Aldrich, L2880) was added for 48 h to damage the HCs. D-gal (Sigma-Aldrich, G0750, 10 mg/mL) was used to induce mimetic aging in the cochlear explants.
Ratcol
Manufactured by Advanced BioMatrix
RatCol is a purified Type I collagen derived from rat tail tendon. It is suitable for cell culture and tissue engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
D gal, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
Deuterium oxide, by Thermo Fisher Scientific (1 mentions)
Methacrylic anhydride, by Merck Group (1 mentions)
2 4 6 trinitrobenzenesulfonic acid solution tnbs, by Merck Group (1 mentions)
Purecol, by Advanced BioMatrix (1 mentions)
3 protocols using ratcol
1
Cochlear Explant Culturing and Damage Induction
2
Cochlear Explant Otoprotection Assay
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Neonatal (P3) mice were sacrificed by cervical dislocation and soaked in 75% alcohol, and the inner ear tissues including the cochlea were dissected using scissors and placed in pre-cooled sterile HBSS. The volute was opened under a microscope, and the spiral ganglion and vascular streaks were removed. The cochlea was then placed face up in a four-well dish that had been previously covered with RatCol (Advanced BioMatrix, 5153). Finally, DMEM-F12 medium containing 10% FBS was added, and the cochleae were cultured in an incubator. After culturing for 12 h, fasudil (0.01 μM) was added for 12 h. The cochleae were then co-treated with neomycin (0.5 mM) for another 12 h. After fasudil and neomycin were removed, the tissues were cultured for an additional 24 h. The number of explants in each treatment group was not less than three, and each experiment was repeated more than four times.
3
Characterization of Collagen Biomaterials
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PureCol, bovine hide type I collagen solution, 3.1 mg/mL (bovine), and RatCol, rat tail type I collagen solution, 4 mg/mL (rat), were purchased from Advanced BioMatrix (Carlsbad, CA). HumaDerm, human skin type I collagen lyophilized (human), was purchased from HumaBiologics, Inc. (Phoenix, AZ). Methacrylic anhydride (2000 ppm topanol A as inhibitor, 94%), collagenase (Clostridium histolyticum, 1873 U/mL), and 2,4,6trinitrobenzenesulfonic (TNBS) acid solution (5% w/v) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and Irgacure (I-2959) were also purchased from Advanced BioMatrix (Carlsbad, CA). Deuterium oxide (99.9 atom % D), deuterium chloride for NMR (20 wt % solution in D 2 O, 100.0 atom % D), and all other chemicals and reagents were purchased from Fisher Scientific (Waltham, MA) unless stated otherwise.
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