Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

Culture immortalized multipotent otic progenitor, or iMOP, cells in media.

Growth factors in the media facilitate iMOP proliferation and colony formation, creating otospheres. 

Collect the otospheres and let them settle by gravity.

Remove the media and add differentiation media lacking growth factors. Plate the otospheres.

The absence of growth factors initiates iMOP differentiation into sensory epithelial cells.

Collect the differentiated otospheres and let them settle.

Remove the media and add a fixative to preserve cellular morphology.

Next, add a detergent to permeabilize cellular and nuclear membranes.

Add a blocking buffer to prevent non-specific antibody binding.

Introduce primary antibodies targeting a specific differentiation marker.

Add fluorophore-conjugated secondary antibodies targeting the primary antibodies, fluorescently labeled phalloidin to stain actin, and a DNA-binding dye to stain the nuclei.

Mount the otospheres.

Using fluorescence microscopy, visualize the staining to confirm iMOP differentiation.