Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons
Transcription
Begin by removing the brains from 5 to 10 E14 embryos in a Petri dish with cold dissection medium. Use forceps to carefully pull out the skin and skull, and isolate the brains. While working under the stereomicroscope, use dissection forceps to remove the meninges. Then, split the telencephalon in the middle to separate the hemispheres.
To isolate the dorsolateral telencephalon cut along the dorsomedial curve and pallial-subpallial boundary. Transfer the dorsolateral telencephalon to a 2-milliliter tube with dissection medium on ice until all brains are dissected. After microdissection of the dorsolateral telencephalon, place the tissue in a 15-milliliter conical tube.
Then, centrifuge the tube containing the collected tissue and cold dissection medium for 5 minutes at 4 degrees Celsius and 340 times g to precipitate the tissue. After the spin, remove the supernatant with the pipette, and add 1 milliliter of 37 degrees Celsius 0.05% trypsin-EDTA for chemical digestion. Incubate for 15 minutes at 37 degrees Celsius.
Following the incubation, add 2 milliliters of proliferation medium to inhibit trypsin activity. Next, polish the tip of a glass Pasteur pipette over a gas burner or Bunsen burner for a few seconds to slightly narrow the aperture. Aspirate FCS to coat the tip of the pipette. Then, mechanically dissociate the cells with the fire-polished and FCS-coated Pasteur pipette by pipetting up and down while being careful to avoid generating bubbles.
Centrifuge the dissociated cells for 5 minutes at 4 degrees Celsius and 340 times g. Remove the supernatant with a pipette. Then, add 1 milliliter of proliferation medium and resuspend the cells using a pipette. Remove the PBS from the pretreated plate with poly-D-lysine. Then, draw a sign on the bottom of the plate that can be used as a reference to determine the zero point.
After resuspending the cells in proliferation medium for the second time, prepare a 1-to-1 dilution of the cell suspension using a 0.4% trypan blue solution. Load into counting chamber slides and count the number of unstained viable cells. Nonviable cells will be blue. Dilute the cells in proliferation medium to get 106 cells per milliliter. Add 500 microliters of the cell suspension to each well of a 24-well tissue culture plate and incubate cells at 37 degrees Celsius and 5% carbon dioxide.
