In this procedure, an in vitro culture is set up to study how cell migration from the ganglionic eminence to the cerebral cortex is affected by different fractions of the cortical protein extract. First latex microspheres are incubated with individual fractions to prepare sources of slowly released proteins. A ferret brain is dissected out and sectioned coronal into thick slices.
Tissue explan are punched out from ganglion eminences. Three-dimensional cultures are set up containing the microspheres and explants embedded in extracellular matrix. After a period of incubation time-lapse image recording is conducted under a brightfield microscope.
The main advantage of this technique over existing methods like using a chemotactic chamber, is that the movements of individual cells can be observed and quantified. This method can help answer key questions in the fields of development of the nervous system, such as chemotactic and Kama. Kinetic control of motility.
Generally, individuals new to this method will struggle because it is difficult to handle the small tissue explants with the matra gel. To begin gradually thaw the frozen matra gel on wet ice to ensure that the temperature remains low enough to prevent premature solidification of the matrix while the matri gel is thawing. Filter sterilize and then slush freeze 500 milliliters of artificial cerebral spinal fluid or A CSF by placing a plastic flask in the minus 80 freezer.
When a thin layer of ice forms on the walls break it off by shaking the flask, then allow another layer of isoform repeating until a nice slurry is created. Next, prepare neuro basal medium according to the manufacturer's instructions and sterilize by filtration. Throw a point 22 micrometer filter membrane incubate at 37 degrees Celsius until ready for use to absorb the protein fraction onto the fluorescent latex microspheres or beads for diffusion into the matrigel, add 10 microliters of the beads to EOR tubes separately containing 100 to 200 microliters of each of five different protein fractions.
Incubate in the dark for 30 minutes at room temperature on a shaker. After the 30 minute incubation, spin the beets down using a benchtop centrifuge at maximum speed for one hour. After deeply anesthetizing the animal with uol, dissect out the whole brain and submerge it in the previously prepared ice cold A CSF.
Fully separate the hemispheres using a scalpel and slice Corona at 500 micrometers. Using a tissue chopper or similar device collecting the slices into a small Petri dish containing ice cold A CSF and sitting on wet ice. Using a dissection microscope, identify slices containing the ganglionic eminences or ge, and trim away regions of cortex that may interfere with subsequent steps.
Press a Harris unicorn bore in the area close to the ventricular surface of the GE.Retract a core containing predominantly the ventricular zone and expel it with a plunger into the ice. Cold neuro basal medium. Transfer a batch of implants from the medium into a 0.5 milliliter drop of ice cold mat gel placed in a separate dish.
Taking care to have the explan fully immersed and avoiding bubbles. Keep the dish on wet ice working in a tissue culture hood. Prepare culture.
Grade six well plates by dotting each well in its center with two microliters of cold matri gel. Using a 20 microliter pipette tip and placing an autoclave round 18 millimeter glass cover slip on each drop. Remove the supernatant and resuspend the beads in 10 microliters of neuro basal medium using a vortex or add 30 microliters of the ice thaw matrigel to the suspension.
Mix well and place on ice. With the eight of the dissecting microscope, place one to two microliters of the bead suspension using a 20 microliter pipette and let it set for one to two minutes, being sure not to let it dry out. Pick up two x explan from the matri gel drop and place them in the desired vicinity of the bead deposit.
Point five to one millimeter away, symmetrically and on opposite sides. Let's set for no longer than five minutes at room temperature. Cover the beads in the X eggplants with a flat layer of about 30 microliters of matrigel and place in a 37 degree Celsius tissue culture incubator for 15 minutes after incubation at two milliliters of neuro basal medium, and then place back in the incubator for three to four days.
Monitoring migratory activity daily after incubation, choose cover slips of interest with implants that have generated a radially uniform cloud of cells. Place chosen cover slips into a fresh culture plate and incubate overnight. Before imaging, use a sterile spatula in a tissue culture hood to move the cover slip in order to release any micro bubbles from under the cover slips.
Fill the space between the wells with water to prevent drying and carefully transfer the plate to the microscope. Using imaging software, define an imaging scheme. Being sure to take into consideration the 3D nature of the culture by collecting a z plane series.
Once the parameters are set, initiate time-lapse. Imaging representative, time-lapse recording of a ganglion, eminence explan generating migrating cells is shown here. Cells migrate radially out of the tissue.
Their attraction to the protein source is variable depending on the nature of the source. In this higher power movie, individual moving cells can be easily traced. As indicated by arrowheads, many cells are seen branching.
A few of these are indicated with arrows. Tracing of individual cells yields positional information that can be further analyzed. Example plots show a differential response between fractions A and D as evidenced by distinct trajectories of cells leaving the explan Once mastered.
This technique can be done in four hours if it is performed properly. While attempting this procedure, it's important to remember to keep mat gel on ice. After watching this video, you should have a good understanding of how to prepare a slow release source of proteins, set a three dimensional tissue culture and conduct time lapse imaging in order to observe and analyze movements of individual cells.
