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- Aspirate medium from a 6-well plate and wash with PBS Ca++/Mg++ free.
- Incubate for 30 min in 1.5 mg/ml Collagenase IV in DMEM:F12 at 37°C.
- Using a cell scraper, remove the colonies and transfer to a 15ml tube.
- Rinse each well with 1ml growth medium and add to the 15ml tube.
- Allow colonies to sediment for 10 min, tapping occasionally to dislodge colonies from the side of the tube.
- Aspirate the supernatant.
- Resuspend gently in 5ml growth medium.
- Allow to sediment for 10 min and aspirate supernatant.
- Resuspend in 10ml growth medium and divide evenly between 2x 15ml tubes.
- Allow to sediment for 10 min and aspirate the supernatant.
- Using a 1ml barrier tip and Gilson, add 1ml differentiation medium and lightly triturate. Differentiation medium should be optimized for the target cell type, but common differentiation media are normal growth medium without bFGF (KSR) or 10% FBS/DMEM (FBS).
- Add a further 6ml of differentiation media and plate in a 60mm suspension culture dish (Corning Ultra Low attachment dishes work best for FBS EBs).
- Change media every 2 days by sedimentation as described above. Resuspend in 7ml differentiation medium.
- Culture for 8 days. Time of differentiation should be optimized for the target cell type.