Dissociation of NSCs and Neurospheres with StemPro® Accutase®
Dissociation of adherent human or rat NSCs grown in StemPro NSC SFM on 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 20uL 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 4 to 6% CO2 in air.
Note: Plating efficiency of 1x106 cells/60mm dish is optimal for the culture system of StemPro® NSC SFM with CELLStart™.
Dissociation of human or rat neurosphere cultures grown in StemPro® NSC SFM
- Remove neurosphere cell suspension from culture dish and transfer to a 15 mL conical tube.
- 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.
- Gently aspirate medium leaving the neurospheres at the bottom of tube with approximately 100 μL of media remaining.
- Resuspend neurospheres in 5 mL DPBS (w/o calcium and magnesium, Cat. No 14190).
- 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.
- Gently aspirate DPBS leaving the neurospheres at the bottom of tube with approximately 100 μL of DPBS remaining.
- Add 1mL of Accutase® to the neurospheres and incubate 10 minutes at room temperature.
- Using the proper sized pipette tip (i.e.1000 μl), pipette up and down until all the neurospheres are in a single cell suspension.
- Add 4mL of fresh medium to the tube.
- Centrifuge the cells at 200g for 4 minutes.
- Gently aspirate the supernatant.
- 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.
Note: 200,000 cells/mL of cell density can be used for cells in StemPro® NSC SFM