To isolate neuro stem cells or NSC's from the dissected neuron subventricular zones or SVZ's, cut each SVZ into four to five small pieces to facilitate the disaggregation of the tissue. Using a sterile, plastic pasteur pipette, transfer the chopped SVZ's to a 15 milliliter centrifuge tube while ensuring no fragments are left in the plate. Let the tissue sediment at the bottom of the tube before removing the remaining DPBS.
Add 500 microliters of filtered containing enzymatic mix for the two SVZ's per brain, and incubate the tube in a water bath at 37 degrees Celsius for 30 minutes. After incubation, add five milliliters of control medium pre-warned at 37 degrees celsius to each sample. Centrifuge the sample at 300 G for five minutes.
Carefully remove the supernatant using a micro pipette or a vacuum pump. Using a fire polished pasture glass pipette, add one milliliter of control medium to the pellet. Mechanically disaggregate the pellet carefully by pipetting up and down 10 to 20 times until a homogeneous cell suspension is obtained.
Then, add four milliliters of control medium, and mix by inversion to wash the cells. After centrifuging at 300 G for five minutes, homogenously re-suspend the resulting pellet in one milliliter of complete medium by pipetting up and down 10 times. After diluting one part of an aliquot of the cell suspension with one part of trypan blue, count the cells using a neubauer chamber under the microscope.
Ensure cellular disaggregation at the single cell level before seeding the cells equally in eight wells of a 48 well plate in a final volume of 500 microliters of complete medium. Incubate the cells for five to seven days to let the primary neurospheres form.
