This article details a protocol for isolating retinal ganglion cells from murine retinae using fluorescence-activated cell sorting (FACS). The method involves dissociating retinal cells, blocking non-specific binding, and labeling cells with antibodies for 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 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.
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