The overall goal of this procedure is to visualize the migration of adult derived neural precursor cells in the presence of a direct current electric field. This is accomplished by first isolating and culturing subependymal neural precursor cells from the adult mouse. The next step is to prepare Galvan Axxis chambers and transfer the neural precursor cells onto the chambers.
The final step is to apply a direct current electric field across the Galvan Axxis chamber and perform time-lapse imaging to observe the neuro precursors migration. Ultimately, the time-lapse imaging microscopy captures the Galvan tactic properties of neuro precursors in their differentiated and undifferentiated states. Though this method can provide insight into neuro precursor cell galvan oasis, it can also be applied to other systems, such as a mouse stroke model for the recruitment of neuro precursors to lesion sites to enhance neuro repair.
Demonstrating the procedure will be Rob Pilley Post a graduate student from my laboratory. When beginning this procedure, NPCs must have been in culture for seven days, so that free floating primary neurospheres are ready to harvest. When required, begin preparing three Galvan Axxis chambers by placing three square glass.
Number one, cover slips in a bottle of six normal hydrochloric acid overnight. Early on the next day, set up the matra gel to thaw slowly on ice. After four hours, it will be ready to use to build the chamber.
First, use a diamond tip glass cutter to cut six rectangular strips of glass from new square. Number one, cover slips. Transfer the acid washed square slips and rectangular slips into a laminar flow hood.
Wash the glass strips with 70%ethanol followed by tissue culture grade autoclave water. Allow them to air dry or dry them on lint-free paper. Next, apply vacuum grease to the perimeter of one surface of each square glass slide and seal them to the base of 60 millimeter plastic.
Petri dishes. Apply vacuum grease on one side of the rectangular glass strips and seal them to opposite edges of the square. Glass slides to create squares with a central trough at the center of each dish.
UV sterilize these modified glass squares or chambers for at least 15 minutes in a hood. Next pipette 250 to 300 microliters of polyol lysine onto the central trough of the chambers. Then incubate the chambers at room temperature for two hours, approximately 15 minutes before the incubation ends.
Prepare the matri gel solution after two hours, aspirate the polyol lysine and wash the central troughs with one milliliter of autoclave water. Then pipette 250 to 300 microliters of Matri gel solution onto the central troughs and incubate the chambers at 37 degrees Celsius for an hour. After the incubation, aspirate the matrigel solution.
Then gently wash the central troughs with one to two milliliters of SFM. Now pipette 100 microliters of E-F-H-S-F-M or F-B-S-S-F-M onto the central troughs in preparation for the cultured cells. Next, pipette four to five milliliters of the neuros sphere containing culture into a new 60 millimeter Petri dish.
Then place the Petri dish on the stage of the counting microscope under the five x viewing objective. Use a P 10 pipette to gently transfer five to eight whole neurospheres onto the central trough of each chamber. Up to four.
Neurospheres may be transferred at once, but do not dissociate them with the neurospheres in the central troughs. Carefully spread them around without disrupting the matrigel substrate. Then add an additional 150 microliters of E-F-H-S-F-M or F-B-S-S-F-M onto the central troughs.
Now culture, the cells in a humidified incubator. After 17 to 20 hours in culture, undifferentiated NPCs will be available after 69 to 72 hours. In culture, differentiated NPCs will be available.
Under accounting microscope, select the chamber with the best cells for live cell imaging migration analysis. The cells should be round nearly completely dissociated from their neurospheres and have not yet, or only just begun to form processes in a hood. Prepare a chamber cover.
Wash a square glass cover slip with 70%ethanol, then with autoclaved water and apply a strip of vacuum grease on two parallel edges. Now cover the chamber. First, aspirate the culture media from the central trough, and then quickly seal the cover, slip onto the rectangular walls like a roof.
Next, using capillary action, load 100 microliters of fresh media into the central trough. Now use vacuum grease to create borders for pools of culture media. On each end of the central trough at the live cell imaging system, have prepared agros gel tubes, silver, silver chloride electrodes, and two 60 millimeter petri dishes to use.
As culture media reservoirs, allow the chambers to rest within the 37 degrees Celsius 5%carbon dioxide environment for 20 to 30 minutes. During this time, prepare the lids of the two empty Petri dishes and the lid of the Galvan Axxis chambers Petri dish by drilling holes into them with a Dremel or tool pipette, 1.5 to two milliliters of media onto either side of the central trough and add seven to eight milliliters of SFM into each empty Petri dish. Place one Petri dish on each side of the chamber's central trough and place one electrode into each dish.
Bridge the gap between the chamber and the Petri dishes to establish electrical continuity. With the Agros gel bridges, connect the electrodes to an external power supply with an M meter in series to measure electrical current and turn on the power supply. Use a vol meter to measure the strength of the electric field directly across the central trough.
Adjust the output of the power supply as needed. Now initiate the time-lapse module on the imaging system. In the Axio vision software Select multidimensional acquisition and click see.
For channels, select channel six, click T for time lapse. Set the time-lapse interval settings to one minute, set the duration to eight hours in the settings before, after time point fields, select the before and after time point. Now click start and allow the experiment to run for the desired amount of time after completion of the assay, carry forward with IHC analysis.
Kinematic analysis revealed that in the presence of a 250 millivolt per millimeter DCEF undifferentiated NPCs exhibit, highly directed and rapid galvan axxis towards the cathode at this field strength, greater than 98%of undifferentiated NPCs migrated for the entire six to eight hours for which they were imaged. In the absence of A-D-C-E-F. Random movement of the cells was observed.
Since dead cells do not migrate, this suggests that they remain viable during this period. In the absence or presence of A-D-C-E-F, differentiated phenotypes underwent negligible migration, both in the presence and the absence of A-D-C-E-F subsets of differentiated cells. Extended processes that tended to align perpendicular to the direction of the DCEF, but no noticeable cell body translocation was observed.
Immunostaining verified that NPCs maintained positive expression of the neural precursor marker nest in after six hours of DC EF exposure. In the assays involving NPCs induced to differentiate into mature phenotypes, the majority of cells express the mature astrocyte marker glial fibrillary acidic protein or GFAP after six hours of DCEF exposure. Following this procedure, one can acquire insights into the cellular mechanisms that are involved in transducing the direct current electric field into cell motility by using genetic knockout models.
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