Protocols: Phoenix ANPGs are isolated as primary cells from the A/J mouse cochlea at P3-P7, a developmental stage characterized by SGN spontaneous spiking (Tritsch and Bergles, 2010) and ensheathment with glial cells (Romand and Romand, 1990).. Spiral ganglion cells isolation and in vitro culture.Collection of mouse inner ear spiral ganglion cells was done as previously described in Rousset et al, 2020 (10.3389/fncel.2020.599152 ). Day 3-7 postnatal A.B6-Tyr+/J (Stock No: 002565, Jackson; carrying the wild type allele of Tyr in a A/J strain genetic background (Stock No: 000646) and C57Bl6/J (Stock No: 000664) pups were used. They were euthanized by decapitation. The head surface was sterilized with 70% Ethanol and was sectioned along the mid-sagittal plane. Brain and brainstem were removed and both half-skulls were immersed in 2.5 ml of ice-cold Hanks' Balanced Salt Solution (HBSS) in a 35 mm Petri dish. The cochleae were removed under a stereo microscope (Nikkon, Japan). The cartilaginous cochlear capsule was opened and the stria vascularis and the organ of Corti (OC) were removed in one piece from base to apex using 5.5 forceps. The modiolus (with the spiral ganglion cells) was then transferred into 1.5 mL Eppendorf tube with ice-cold HBSS. Tissue dissociation was done by enzymatic digestion followed by mechanical trituration. The modiolus was treated with Accutase (StemPro™ Accutase™ Cell Dissociation Reagent) for 15 minutes, the reaction was stopped by adding proliferation media: DMEM:F12 with 15mM HEPES buffer and 2mM L-glutamine supplemented with 1X N2 and B27 supplement (Thermo Fisher), 1X Penicillin streptomycin (100U/ml) (Thermo Fisher), in presence of bFGF (10ng/ml, ProSpec), IGF1 (50ng/ml, Cell Guidance Systems), Heparan sulfate (50 ng/ml, Sigma Aldrich) and EGF (20 ng/ml, Cell Guidance Systems) and mechanical trituration was performed with P1000 (10-15 up and downs). Following Accutase removal with a centrifugation step, the cell suspension was filtered with a 70µm filter to eliminate undissociated tissue or bony parts and select small cluster of spiral ganglion cells. Isolated cells were maintained in culture with proliferation media in ultra-low attachment 6 well plates (Corning, Sigma Aldrich). Passaging of spiral ganglion cells-derived neurospheres was done twice a week using enzymatic digestion with Accutase™ followed by mechanical trituration as previously described RNA extraction was performed using a Qiagen RNA extraction minikit (Qiagen), according to the manufacturer's protocol. TruSeq ribodepleted stranded mRNA was applied to all samples to eliminate ribosomic RNA
ENA-FIRST-PUBLIC
2022-08-31
ENA-LAST-UPDATE
2022-08-31
External Id
SAMEA110178670
INSDC center alias
The Inne ear and Olfaction laboratory, Department of Clinical Neurosciences, Medical Faculty, University of Geneva
INSDC center name
The Inne ear and Olfaction laboratory, Department of Clinical Neurosciences, Medical Faculty, University of Geneva
INSDC first public
2022-08-31T00:13:27Z
INSDC last update
2022-08-31T00:13:27Z
INSDC status
public
Submitter Id
E-MTAB-11869:Sample 10
age
5
broker name
ArrayExpress
cell_type
auditory neuroprogenitor
cohort
Phoenix ANPGs
common name
house mouse
developmental stage
juvenile stage
disease
normal
genotype
wild type genotype
organism part
cochlea
sample name
E-MTAB-11869:Sample 10
strain
A.B6-Tyr+/J