Direct Reprogramming of Hematopoietic Progenitor Cells into Neural Stem Cells
Transcript
When the cells are 30 to 50% confluent, aspirate the supernatant and reserve, and then add 1 milliliter of neural progenitor medium to each well. To make a backup plate using the spheres in the supernatant, centrifuge the reserve supernatant at 170 times g for 10 minutes.
Then, aspirate the supernatant and resuspend the pellet in neural progenitor medium. Plate in a matrigel-coated 24-well plate; change the medium in each plate of cells every other day until the cells reach 60% to 80% confluence, usually after one week.
Once the cells reach the required confluence, aspirate the supernatant, and add 1 milliliter of neural stem cell medium to each well. Then, dissociate the cells by using a cell scraper followed by very gentle pipetting.
Next, transfer the cells from one well of the 24-well plate into one well of a 6-well plate. Add another milliliter of medium to the well, and after repeating the procedure for all wells, place the 6-well plate into the 37 degrees Celsius 5% carbon dioxide incubator.
Change the full volume of medium every other day until the cells reach 60% confluence. After each time, dissociate and replate the cells at a 1-to-3 ratio as just shown.
