Dissociation of hESCs with StemPro Accutase

1. Aspirate the medium from culture dish and wash with 4mL of DPBS (w/o calcium and magnesium, Cat. No 14190).


2. Aspirate DPBS and add 2 mL of Accutase to culture dish.


3. Incubate for 2 to 5 minutes at 37 °C until individual single cells start to round up.


4. Gently rinse to remove cells off of the plate’s surface.


5. Transfer cell suspension to 15 mL conical tube.  Gently pipette up and down until cells are in a singe cell suspension.


6. Add 8 mL of medium to rinse any remaining cells off of the dish’s surface and transfer to the conical tube (from Step 5).


7. Take a 20 µL sample of the cell suspension to determine viable cell density.


8. Centrifuge conical tube containing the cell suspension at 200g for 4 minutes.


9. Aspirate supernatant, resuspend in fresh medium and plate on coated dish(s).  Incubate at 36 to 38°C in a humidified atmosphere of 5% CO₂ in air.

Note: Plating efficiency of 0.5–1x10⁶ cells/60mm dish is optimal for the culture system of StemPro hESC SFM with Geltrex.