Dissociation of hESCs with StemPro® Accutase®
Dissociation of hESCs grown in StemPro® hESC SFM on Geltrex™ hESC-qualified BME or CELLstart™ coated dishes
- Aspirate the medium from culture dish and wash with 4mL of DPBS (w/o calcium and magnesium, Cat. No 14190).
- Aspirate DPBS and add 2 mL of Accutase® to culture dish.
- Incubate for 2 to 5 minutes at 37 °C until individual single cells start to round up.
- Gently rinse to remove cells off of the plate’s surface.
- Transfer cell suspension to 15 mL conical tube. Gently pipette up and down until cells are in a singe cell suspension.
- 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).
- Take a 20 µL sample of the cell suspension to determine viable cell density.
- Centrifuge conical tube containing the cell suspension at 200g for 4 minutes.
- 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™.