Dissociation of NSCs and Neurospheres with Accutase®
Dissociation of adherent human or rat NSCs
1. Aspirate the medium from culture dish.
2. 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 single 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 20uL 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 4 to 6% CO2 in air.
Dissociation of human or rat neurosphere cultures
1. Remove neurosphere cell suspension from culture dish and transfer to a 15 mL conical tube.
2. Let neurospheres settle down in the tube (~2 to 5 minutes) before proceeding to Step 3. Alternatively, the cells can be centrifuged at 100g for 1 minute.
3. Gently aspirate medium leaving the neurospheres at the bottom of tube with approximately 100 μL of media remaining.
4. Resuspend neurospheres in 5 mL DPBS.
5. Let neurospheres settle down in the tube (~2 to 5 minutes) before proceeding to Step 6. Alternatively, the cells can be centrifuged at 100g for 1 minute.
6. Gently aspirate DPBS leaving the neurospheres at the bottom of tube with approximately 100 μL of DPBS remaining.
7. Add 1mL of Accutase® to the neurospheres and incubate 10 minutes at room temperature.
8. Using the proper sized pipette tip (i.e.1000 μl), pipette up and down until all the neurospheres are in a single cell suspension.
9. Add 4mL of fresh medium to the tube.
10. Centrifuge the cells at 200g for 4 minutes.
11. Gently aspirate the supernatant.
12. Resuspend cells in fresh medium, transfer to a new culture dish and incubate at 36 to 38°C in a humidified atmosphere of 4 to 6% CO2 in air.