Differentiating Immortalized Multipotent Otic Progenitors into Spiral Ganglion Neurons and Evaluating the Differentiation
Transcription
Take a multi-well plate containing sterile coverslips coated with a cell culture substrate.
Introduce immortalized multipotent otic progenitors, or iMOPs, in a neuronal differentiation medium.
iMOPs are precursors to various inner ear cells, including spiral ganglion neurons, or SGNs, that transmit auditory information to the brain.
Incubate, allowing the cells to adhere to the substrate. The medium induces differentiation of the iMOPs into SGNs.
Add a fixative to induce protein crosslinking, preserving cell morphology, and a detergent solution to permeabilize cellular membranes.
Introduce a blocking reagent to prevent non-specific labeling.
Add fluorophore-conjugated antibodies specific for neuronal differentiation markers and incubate, allowing the antibodies to label the SGNs.
Wash to remove unbound antibodies, and mount the coverslips with a mounting medium containing a fluorescent nuclear stain.
Using fluorescence microscopy, detect the antibody signals to confirm the differentiation of iMOPs into SGNs.
