フローサイトメトリーによる一次マウス網膜神経節細胞（RGCs）の単離

The overall goal of this procedure is to isolate an enriched homogeneous population of primary murine retinal ganglion cells. This method can help answer questions in the retinal ganglion cell field about the mechanisms underlying the declining visual acuity in age populations and in retinal degenerative disease populations. The main advantage of this technique is that it can offer a fast, visible, reproducible, and standardized protocol for the isolation of enriched primary murine retinal ganglion cells.

We first had the idea for this method when Sumana Chintalapudi, who was then a graduate student in my lab and first author on this study, presented at our journal club a 2013 paper by Krishna Murtheadol, and their study clearly demonstrated that the RGC five cell line, which was originally generated as an immortalized rat retinal ganglion cell line, was no longer of rat origin, nor did it possess the RGC phenotype. It became apparent to us that this problem left a large gap in the resources available to the vision research community, including our own lab. Sumana and I reached out Dr.Morales-Tirado and together the three of us developed a new method for isolating live and highly enriched RGCs.

Generally, individuals new to this method will struggle because the goals of this method are to isolate a live and highly enriched cell population that can be either cultured as primary cells, or immortalized as a cell line. Because of this one must be very careful not to damage the cells that one is trying to isolate. In addition, the parameters of cell sorting method must be tailored for the specific cells one is trying to isolate.

Both methods require special skill sets. Demonstrating the procedures will be Dr.Xiang Di Wang, a research associate from Dr.Jablonski's lab, and Mr.Zach Goldsmith, PhD candidate from my laboratory. To enucleate the eye, first insert forceps under the globe of the eye.

Grip the optic nerve and pull up. The globe will be enucleated with the optic nerve intact. Place the eye in a vial of PBS on ice, and enucleate the other eye.

When all of the eyes have been collected, place one eye in a Petri dish of fresh PBS under a dissection microscope, and use forceps to carefully grasp the globe at the base of the optic nerve. Using a sharp 30 gauge needle, puncture the cornea to allow the aqueous humor to drain making it easier to hold the eye with the forceps. Holding the cornea with the forceps, use scissors to make a small incision in the corneal tissue and use the forceps to gently peel away the cornea and the sclera.

When the globe is peeled halfway, use the forceps to roll out the retina and the lens. Place the retina in a small 40 millimeter Petri dish containing PBS and 1%FPS in a biosafety cabinet and wash the retina three times in fresh PBS plus FBS for each wash. When all of the eyes have been washed, place up to 12 retinae onto a sterile 70 micron nylon strainer moistened with PBS and FBS and gently macerate the retinae 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 one milliliter of solution per retina and collect the cells by centrifugation.

Then, resuspend the pellet in PBS plus FBS at a one milliliter of medium per five retinae concentration, if immunolabeling is to be performed immediately. Alternatively, incubate the cells resuspended in neural cell medium overnight at four degrees Celsius in the horizontal position. To immunolabel the retinal cells, centrifuge the retinal cell suspension, and resuspend the pellet in 50 microliters of fresh PBS plus FBS in a fax tube.

Set aside five times 10 to the sixth cells for the unlabeled negative control. Next, block any non specific FC receptor binding in each tube with one microliter of anti mouse CD 16 32 antibody per one times 10 to the sixth 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 formula liter 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 two washes in PBS plus FBS as just demonstrated. Resuspend the pellets in fresh PBS and FBS for counting cells.

To adjust the compensation add three drops of polystyrene microspheres into one sterile fax tube per florafor and one microgram of the respective florafor to each tube. Incubate the microspheres for 15 minutes at room temperature protected from light. Then wash the microspheres in three milliliters of PBS and FBS and carefully remove the supernatant.

Resuspend the microsphere pellets in 250 microliters of PBS and FBS and load the unlabeled sample tube onto the flow cytometer. In the flow cytometer software, select experiment, compensation set up, and create compensation controls. Add the florafor specific controls from the displayed list and click OK.Verify the forward scattered and side scattered light, and gate the initial population.

Then install the negative control tube onto the cytometer and click load. Verify that the population of interest or P1 is displayed and select the population. Right click the P1 gate and select calculate compensation.

Then click record data. When the recording is finished, click unload to remove the tube and load the next control tube to adjust the compensation controls. When all of the control samples have been recorded, create a forward versus side scatter plot for the first experimental tube, and gate the P1 population.

Next, open a side scatter height versus side scatter width pseudo color plot and gate the single cells. Using the single cells, create a forward scatter height versus forward scatter width plot, and gate to exclude the dublets. Then generate a CD48 versus CD90.2 pseudo color plot and gate the CD90.2 positive CD48 negative cells to eliminate the monocytes, followed by a CD57 versus CD15 pseudo color plot to eliminate any amacrine cell contaminants.

Now define the populations to be collected for the sample in the sort layout sheet, and sort the cells. At the end of the sort, use a 2.5 times 10 to the fourth cell aliquot to confirm the purity of the isolated cell population. After meshing the retinae in the cell strainer multiple inner retinal cells can be visualized, even though the retinal ganglion cells have lost their signature morphology, due to axotomy during the cell isolation procedure.

The classic dye one positive CD48 negative surface phenotype is not sufficient for identifying and isolating the murine retinal ganglion cells, as these cells express genes associated with amacrine, muller, bipolar, horizontal photo receptor, and retinal pigment epithelial cells. The sorted cells also express retinal ganglion cell associated intracellular markers such as synuclein gamma, BRN3A, TUJ1, and RBPMS, with the intracellular localization of the markers further confirmed by imaging flow cytometry. Some of the sorted cells even begin to exhibit retinal ganglion cell morphology after in vitro cell culture.

Further, quantitative PCR analysis of the highly enriched sorted cell population reveals a many fold increase in the genes coding for retinal ganglion specific intracellular markers. Once mastered, this technique can be completed in less than six hours if it's performed properly. While attempting this procedure, it's important to remember to keep your cell suspension sterile, to block the FC receptors from the cell suspension, to avoid non specific binding of the antibodies, and to discuss your cell sorting details or strategy with the cell sorter operator.

Following this procedure, other methods like QPCR can be performed to answer additional questions about gene expression profiling. Don't forget that working specialized equipment, such as a cell sorter, requires a highly specialized operator. After its development, this technique paved the way for researchers in the vision field to help understand survival and death mechanisms in retinal ganglion cells, for the development of novel therapeutics for vision preservation.

After watching this video, you should have a good understanding of how to isolate an enriched homogeneous population of primary murine retinal ganglion cells.