The overall goal of this procedure is the simultaneous generation of neural precursor cell cultures as either adherent monolayers or neurospheres from the subventricular zone and dentate gyrus of individual adult mice. This is accomplished by first removing the brain of individual adult mice. The second step is to microdisect the sub ventricular zone and dentate gyrus regions.
Next, the tissue is dissociated into a single cell suspension. The final step is to culture the precursor cells as either adherent monolayers or neurospheres. Ultimately, the neuros sphere assay and the adherent monolayer culture system are valuable tools to determine the potential for proliferation or differentiation in adult neural stem cells.
In vitro, This method allows us to address a couple of very interesting questions in the adult neurogenesis field. With it, we can look at cells from individual animals and compare them, and this is of interest in the context at looking at individual differences, or, for example, at differences between individual transgenic or knockout mask compared to their controls. Tara Walker, a senior scientist in my group, would present this method Before brain dissection.
Prepare the fire polished pipettes with medium and small boars by rotating the glass pasture pipettes in a flame until the edges become rounded. Then sterilize them in an autoclave. First, transfer the brain to a 10 centimeter plastic Petri dish containing PBS.
Place a Petri dish with the brain under a dissecting microscope at low magnification with the brain positioned on its ventral surface. Subsequently, use the fine curved forceps to remove the olfactory bulbs while holding the cerebellum. Next, rotate the brain onto the dorsal aspect.
Make a coronal cut through the brain at the level of the optic chiasm with a scalpel to microdisect the SVZ. Place the rostral portion of the brain against the dish so that the cut coronal surface faces upward. Under a higher magnification, remove and discard the septum.
Then dissect the SVZ, which is a thin layer of tissue surrounding the ventricle by placing the tip of one blade of the fine curved forceps in the lateral corner of the lateral ventricle. Immediately under the corpus callosum and the other blade, approximately one millimeter into the tissue immediately adjacent to the ventricle. Press down the forceps towards the base of the dish and towards the ventral aspect of the ventricle to remove a small triangular piece of tissue.
After that, place the dissected SVZ into a Petri dish on ice to microdisect the DEG place, the coddle portion of the brain in the Petri dish and cut along the longitudinal fissure using a scalpel under a dissection microscope. Remove the cerebellum and the dye cephalon. Refocus the microscope so that the borders around the DG are now visible.
To remove the dg, insert the tip of a 27 gauge needle and slide along the border between the DG and amen's horn. Then free the DG from the surrounding tissue using the fine forceps For SVZ tissue dissociation. Mince the tissue using a scalpel blade for a minute until no large pieces remain.
Then resus. Suspend the mince tissue with one milliliter of Prewarm 0.05%trips in EDTA. Afterwards, transfer it to a 15 milliliter tube and incubated for seven minutes in a water bath set to 37 degrees Celsius.
To stop the enzymatic reaction, add one milliliter of trypsin inhibitor containing DNA swan and mix the contents by flicking the tube. Next pellet the suspension by centrifugation at 300 Gs for five minutes and a scar. The sennett after that resuspend the pellets in one milliliter of growth medium and associated by gly.
Pipetting up and down approximately seven to 10 times using a P 1000 pipette. Then add the growth medium to a total volume of five milliliters and pass the cell suspension through a 40 micrometer sieve to remove debris and unassociated tissue clumps. Subsequently centrifuge it at 300 Gs for five minutes.
Discard the supinate and reus. Suspend the resulting pellet in 200 microliters of growth medium for DG tissue dissociation. Mince the tissue using a scalpel blade for approximately one minute until no large pieces remain.
Next, transfer the mince tissue into the prewarm PDD enzyme mix. Incubate it for 20 minutes at 37 degrees Celsius and mix it by inverting the tube every three to five minutes. Next, dissociate the tissue mechanically by pipetting it up and down gently 10 times using a medium bore fire polished pasture pipette, then incubated for a further 10 minutes at 37 degrees Celsius and mix it by flicking the tube every three to five minutes.Further.
Dissociate the tissue mechanically by pipetting it up and down gently 10 times using a small boar fire polished pasture pipette. Afterwards, centrifuge it at 130 gs for five minutes. Subsequently, remove the supinate and resuspend the pellet in one milliliter of buffer solution.
Make up to 10 milliliters with buffer solution. After centrifugation at 130 GS for five minutes. Remove the S supernatant and resuspend the pellet in five milliliters of 20%per call.
Then centrifuge it at 450 GS for 15 minutes. Remove the snat and resuspend the pellet in 10 milliliters of buffer. Finally, centrifuge at 130 GS for five minutes and resuspend the pellet in 200 microliters of growth medium for adherent cultures plate 200 microliters of cells in each single well of A PDL Lamin ENC coated 96 Well plate for sphe cultures dilute it with growth, medium to 20 milliliters and plate 200 microliters per well over an uncoated 96 well plate then incubate at 37 degrees Celsius with 5%CO2 after six to 12 days count and measure the diameter of the neurospheres using an IP gradi fitted to an upright light microscope.
This figure shows that the adult mouse precursor cells can be cultured as adherent monolayer cultures or as neurospheres in the SVZ and dg. Significantly more neurospheres are seen to be generated from the SVZ compared to the DG of individual mice and the SVZ and DG precursor cells also respond differently to in vitro depolarization to confirm long-term potentiation. Neurospheres are shown to have expanded for 10 passages.
Neurospheres can be differentiated into beta three tubulin positive neurons as shown in red GFAP positive astrocytes in green, O four positive oligodendrocytes in red and map two AB positive neurons in red Following this procedure. Other methods such as packaging the cells or neurospheres over several generations or differentiation of the neurospheres followed by histological analysis can be performed. This will help answer further questions such as multi potentiality and long-term potential.
