Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Nestin-expressing progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

The overall goal of this procedure is to isolate specific cell populations from the developing cerebellum. This is accomplished by first harvesting cerebella from neonatal mice carrying fluorescent proteins. The second step of the procedure is to obtain live tissue sections using a vibrato.

The third step is to microdisect the external germinal layer of the cerebellum under a fluorescent dissecting microscope. The final steps are to dissociate the tissue into a single cell suspension and collect cells by fluorescent activated cell sorting. Ultimately, results can show that specific cell populations can be isolated from the cerebellum.

The main advantage of this technique over existing methods, such as laser capture, micro dissection, is that cells are living and can be cultured. For further experimentation For this procedure, sterilize the surgical tools using an autoclave. Get fresh or reheated 3%low melting aros ready to use in a 37 degrees Celsius water bath.

Make up the required solutions for the papayan based cell dissociation. Prepare fresh NBB 27 culture media. Then filter, sterilize, and store in an incubator.

Prepare the fax buffer and prepare the poly de lycine cover slips. First coat autoclave cover slips with 100 micrograms of poly de lycine per milliliter of sterile water. Then incubate the cover slips for two hours at 37 degrees Celsius or overnight at room temperature.

Later, wash the cover slips with distilled water followed by NBB 27 media before using the cover slips. Dry them completely. This will take at least an hour before beginning.

Prepare the dissection area first. Wipe it clean with 70%ethanol. Then cover it with an absorbent pad.

Second, remove the tools from the sterilization pouch and position them for use. Third, load a dish with ice cold sterile DPBS with 1%pen strip and put it on ice. Dissect out the brain of a P four pup that expresses fluorescent markers.

Then transfer it to the prepared dish of DPBS. Use forceps to carefully separate the cerebellum from the rest of the brain. These cerebella express the markers math one, GFP, and nest in CFP in which the EGL expresses GFP and the molecular layer is positive for CFP.

Next, fill a two centimeter cubed embedding mold with the agro solution. Make sure that it is not any warmer than 37 degrees Celsius. Pick up a cerebellum with a perforated spoon and pad it dry with a gentle dabbing of tissue.

Then using forceps, set it into the mold. Use a needle to get the cerebella into vertical orientations. Once about four are loaded into the mold.

Place the mold on ice so it will quickly solidify Using a razor blade. Trim the block to reduce the sectioning time. Then glue the block to the plate oriented so the cerebella are vertical.

Load the filling tray with ice cold DPBS with 1%pent strap and put ice under the tray so it stays cold. Now section the blocks with a vibrator. Cut and collect 600 micron sections.

Remove them from the ice tray using a perforated spoon and transfer them to A-D-P-B-S filled dish on ice. Under fluorescent dissecting microscope. Examine the slices as quickly as possible.

Carefully separate the EGL from the rest of the cerebellar section. Use fine forceps to dissect between the CFP positive molecular layer and the GFP positive EGL. Be very conservative during microdissection to avoid contamination from adjacent regions.

Transfer the GFP positive EGL tissue to a new DPBS filled dish on ice. Avoid including any molecular layer tissue as this will contaminate the culture with nesting expressing Bergman glia. Next, remove the agros surrounding the isolated EGL tissue.

The EGL can now be dissociated using a paan based protocol for a single cell suspension. When this is completed, resus suspend the cells in fax buffer. Proceed with collecting the CFP cells using fax.

Sort them using a high speed cytometer and a CFP filter and pull them into ice cold fax buffer. About 100, 000 NPS should be obtained per P four cerebellum. Then centrifuge the pooled cells at 300 Gs for five minutes.

Make immediate use of the cells to culture them resus. Suspend them in prewarm NBB 27 media. Then load them onto the poly D lysine coated cover slips.

Cell bella slices were prepared from P four math one GFP nest in CFP mice. Cerebellar EGL contained mainly GFP expressing GMPs and could be easily separated from the molecular layer enriched in CFP expressing glia Facts analysis showed that more than 85%of the cells in the EGL were conventional GNPs. NEPs were isolated from the deep parts of the EGL by microdissection, followed by facts.

These cells accounted for about 5%of the cells in the EGL four days of culturing. The facts isolated NPS gave rise to beta tubulin expressing neurons. This is characteristic of lineage restricted neuronal progenitor cells.

While attempting this procedure, it's important to remember to work quickly and keep the tissue on ice as much as possible.