Begin by placing a sacrificed pregnant mouse on its back on a dissection board. Using forceps, pinch the abdominal midline. With a pair of sharp scissors, cut the abdomen through the skin and the peritoneum from the genitalia to the rib cage over the midline.
Remove the uterus containing the embryos and place it immediately on ice. Carefully cut through the yolk sac on the placental sides and remove the embryos. Then, place the decapitated embryonic heads into an iced dish containing calcium and magnesium-deficient HBSS.
Place the head into a 35-millimeter dish facing left. Pierce one eye with the edge of the forceps, firmly holding the chin with the other. Gently tear the scalp skin from the nape along the midline towards the snout.
Use the angled forceps to enter through the white oval spinal cord and crack the skull open along the midline, exposing the brain. Gently peel the skull away from the sides. Then, lift the brain out of the skull and dispose of the skull.
Use forceps to remove the meninges. Place the brains in a bijou containing two milliliters of calcium and magnesium-deficient HBSS. Add 250 microliters of 10X trypsin to the bijou.
Triturate the brains by shaking the bijou and then incubate for 15 minutes at 37 degrees Celsius. Next, add two milliliters of soybean trypsin inhibitor to each bijou. Shake to disperse the inhibitor evenly.
Without centrifuging, transfer two milliliters of the supernatant from each bijou into a 15-milliliter centrifuge tube. Using a 19-gauge needle attached to a five-milliliter syringe, aspirate the suspension twice to triturate the remaining cells in the bijou. Repeat aspiration twice with a 21-gauge needle.
Using a 23-G needle, transfer the cells from the bijou into the 15-milliliter centrifuge tube containing the cell supernatant and centrifuge at 200 G for five minutes at room temperature. Using a five-millimeter serological pipette, transfer all the supernatant into a new 15-milliliter centrifuge tube without disturbing the pellet. Repeat centrifugation.
Add 10 milliliters of the plating media to a tube containing the combined pellets from both centrifugation steps and mix well to create a whole suspension. Stain the cells with trypan blue and count using a hemocytometer or a cell counter. Plate the cells by adding the required volume of the murine E17 embryonic brain cell suspension into a well plate.
Incubate the cells at 37 degrees Celsius under 5 to 7%carbon dioxide for two to four hours. After removing the media, add new differentiation media to each well. Press down any floating cover slips using a sterile pipette tip.
Maintain cultures by removing part of the supernatant and replacing it with fresh differentiation media three times each week. Cultures were visualized by immunofluorescence staining for NG2 and nestin as developmental markers, SMI31, MBP, and NeuN as neuronal markers, and CNP, GFAP and Iba1 as glial markers Infection of cultures with neurotropic Semliki Forest virus affected the oligodendrocytes and the neurons. qRT-PCR investigations of the infected cells demonstrated upregulation of the chemotactic cytokine Ccl5 mRNA in cultures treated with interferon beta.
ELISA studies displayed increased Ccl5 in the supernatant, thus displaying a correlation between mRNA and protein expression. Flow cytometric studies of single-cell suspension showed that 70%of the cells remained viable. A large number of microglia, neurons, and astrocytes were present, while oligodendrocytes were the least abundant.
