Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting
Transcription
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.
