To perform passage of neurospheres obtained from neuro stem cells isolated from neuron subventricular zone, collect the medium containing the neurospheres from the multi-well plates and transfer them to a 15 milliliter centrifuge tube. Centrifuge the neurospheres for five minutes at 300 G.After removing the supernatant, add 200 microliters of enzymatic solution to the pellet and gently tap the bottom of the tube to dislodge it. Incubate for 10 minutes at room temperature to facilitate the neurosphere dissociation.
Then add one milliliter of control medium to halt the enzymatic reaction. Mechanically dissociate neurospheres with a P 1000 micro pipette by pipetting up and down 10 to 20 times. Add an additional volume of four milliliters of control medium and mix by inversion to wash the cells.
Centrifuge the cells for five minutes at 300 G before removing the supernatant. Add one milliliter of complete medium and resuspend the pellet to homogenize the cellular suspension 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 cell suspension using a Neubauer chamber.
To expand the culture, seed cells at a density of 10, 000 cells per square centimeter using complete medium in an appropriate culture plate or flask.
