Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting

Place a murine retina on a moistened cell strainer and macerate in a circular motion to separate the retinal cells from the extracellular matrix.

Transfer the strainer to a collection tube.

Add a phosphate buffer and serum solution to the strainer to wash and collect the cells.

Centrifuge to settle the cells. Discard the supernatant and resuspend the cells in a phosphate buffer and serum solution.

Add blocking antibodies that bind to Fc receptors on cells, preventing non-specific binding of target antibodies.

Next, add fluorescently tagged and untagged primary antibodies that bind to specific cell surface markers on various cell types.

Wash to remove unbound antibodies.

Add a fluorescently tagged secondary antibody to bind to the untagged primary antibody.

Wash to remove unbound antibodies.

Perform fluorescence-activated cell sorting, or FACS to capture the distinct fluorescent signals of the labeled cells and sort the retinal ganglion cells from various retinal cell populations.

Place up to 12 retinae onto a sterile 70-micron nylon strainer moistened with PBS and FBS, and gently macerate the retina with the back end of a 10-milliliter syringe plunger using a circular motion. When