Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts
Transkript
One day later, aspirate laminin from the plates, and add 200 microliters of neural induction medium to the wells. Wash the six-well plates containing the colonies to be picked, once with DPBS, before adding 2 milliliters of neural induction medium per well of a six-well plate.
Mechanically pick the cells by scraping around the colonies using a thin needle to get rid of surrounding cells, and picking the colonies using pipette tips mounted on a 200-microliter pipette man set on 50 microliters.
Transfer each colony into one well of a laminin-coated 48-well plate and generate a single-cell suspension mechanically by pipetting up and down 10 times. Add Rho-kinase inhibitor in a final concentration of 10 micromolar to the cells. Grow the cells on the 48-well plate at 37 degrees Celsius, 5% CO2 for two days. Change the medium every second day until the cells reach 80% to 90% confluency.
