Differentiating Embryoid Body Cells into Neural Progenitors Using Retinoic Acid

Begin embryoid body formation by harvesting embryonic stem cells with 0.25% trypsin-EDTA for 2 to 5 minutes at 37 degrees Celsius, 5% carbon dioxide. Add fresh Iscove's Modified Dulbecco's medium or IMDM with 15% FBS to the detaching cells, then transfer the cell suspension to a 15-milliliter tube.

Centrifuge at 160 x g for 5 minutes at room temperature. Then, count the cells using a hemocytometer and prepare a 5 x 10⁵ cells per milliliter suspension in IMDM with 0.5 micromolar retinoic acid. Next, use an 8-channel pipette and 200-microliter tips to plate 120-microliter drops per 100-milliliter Petri dish. Invert the dish and fill the inverted lid with PBS to prevent hanging drops from drying. Culture at 37 degrees Celsius, 5% carbon dioxide for 3 to 4 days.