Isolating Murine Retinal Ganglion Cells Using Fluorescence-Activated Cell Sorting

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 all of the cells have been dissociated, place the strainer over a polypropylene collection tube, and use a P1000 pipette to filter the cells through the strainer into the collection tube.

Rinse the strainer with PBS and FBS, pooling the wash in the collection tube. Add enough PBS plus FBS to bring the final volume up to 1 milliliter of solution per retina, and collect the cells by centrifugation. Then, resuspend the pellet in PBS plus FBS at a 1 milliliter of medium per 5 retinae concentration. Next, block any nonspecific FC receptor binding in each tube with 1 microliter of anti-mouse CD16/32 antibody per 1 x 10⁶ cells for 10 minutes at room temperature.

At the end of the blocking incubation, add the antibody cocktail of interest to the cells with gentle pipetting for a 30-minute incubation on ice protected from light. Then, wash the cells two times in a 4-milliliter final volume of PBS and FBS. Label the cells with an appropriate secondary antibody for another 30 minutes on ice protected from light, followed by 2 washes in PBS plus FBS, and load the sample tube onto the flow cytometer.