Accutase™

The color of your Accutase™ is caused by phenol red, which depends on the pH. During shipment, it is possible that small amounts of CO₂ permeate into the plastic bottle that could change the pH, resulting in a slightly lower pH (turning yellow).

The original pH is 7.3, yet changes to pH 6.8 can occur during transport. This is nothing you need to worry about, as it will not affect the performance of your Accutase™.

Yes, it is still OK to use.

The individual components do not defrost evenly, therefore it is possible that you can observe uneven color distribution within the bottle. Just make sure to mix well by shaking gently (preferably by inverting), once the product is fully defrosted.

Yes, you can use Accutase™ to dissociate cell clumps.

If you should not succeed using Accutase™, you can use Accumax™ instead. It contains the same enzymes but at a higher concentration.

If you defrosted your Accutase™ in a +37 °C water bath and the temperature did not reach +37 °C, it may still be OK to use (reduced activity is possible). If you heated the product to +37 °C, it will definitely have lost its enzyme activity.

Please observe whether your Accutase™ is still useable and discard the damaged product if necessary.

No, this is not necessary.

Before detachment, simply pour off/aspirate the medium and add Accutase™. After detachment, you can simply add fresh medium to your detached cells.

The incubation times are similar to trypsin, you can simply proceed the way you are already used to doing it!

Do not worry about overexposing your cells to Accutase™ – it is very gentle, and your cells shouldn't take any damage, even when exposing them for a prolonged time.

We advise you to determine the optimal detachment/exposure time for your cells individually!

When your Accutase/Accumax™ products arrive, they should be frozen (at least a small ice cube) and have a yellow color that changes to orange when defrosting.

Once unpacked, you can immediately refreeze the product or place it into the fridge for defrosting.

• Never defrost Accutase/Accumax™ in a +37 °C water bath – this will destroy the product!
• Defrost overnight in the refrigerator or place in a tub of cold tap water for ~ 1.5 hours.
• After defrosting, always remember to shake the bottle. The individual components do not go into solution evenly when defrosting!
• Accutase/Accumax™ is stable in the refrigerator for 2 months, therefore aliquotation is not necessary. Yet, refreezing aliquots is possible.

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 20 µL sample of the cell suspension to determine viable cell density.
8. Centrifuge conical tube containing the cell suspension at 200 x g 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.

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 100 x g 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 100 x g for 1 minute.
6. Gently aspirate DPBS leaving the neurospheres at the bottom of tube with approximately 100 μL of DPBS remaining.
7. Add 1 mL 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 4 mL of fresh medium to the tube.
10. Centrifuge the cells at 200 x g 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.