Accutase™ is a mix of proteolytic and collagenolytic enzymes and serves as a direct replacement for trypsin and collagenase applications. It is free of mammalian and bacterial origin and requires no inactivation step during passaging of cells.
When compared to trypsin, Acctuase™ can be used at far lower concentrations than traditional trypsin solutions. This offers a gentle detachment option and is therefore less harmful for your cells (e.g., no damaging of surface epitopes of the cell).
Accutase™ is a direct & gentle replacement for trypsin. Tested with various applications and cell lines.
NO MAMMALIAN ORIGIN
Accutase™ is a non-mammalian, non-bacterial cell detachment solution.
Simply dilute the cell solution after use to stop detachment. Inactivation is not necessary!
Cell detachment is an essential process for the subcultivation of anchorage-dependent cells. As adherent cells grow, the substrate eventually becomes fully occupied. Also, the cell concentration might be too high for the capacity of the growth medium used. This is the time when cells must be split to prevent unwanted cell death by overpopulation.
Another scenario is the aggregation or clumping of cells, where cells must be detached from each other.
Cells usually possess a negative surface charge that is unevenly distributed and dependent on the current physiological state of the cell. In most cases, the cultivation surface has a negative charge and adhesion is believed to be mediated by protein bridges or ionic interactions (e.g., via Ca2+).
Protein bridges, also called substrate adhesion molecules, have receptors on the cell side, as well as adhesion molecules on the substrate side. There are roughly 30 adhesion molecules, such as collagen, fibronectin, laminin, and vitronectin.
By far the most common way to detach cells is via trypsinization.
Trypsin is a cheap proteolytic enzyme, usually extracted from the pancreas of pigs (porcine trypsin), and has been used successfully for various applications. Also, it can be inactivated by serum, which is very convenient for cell culture.
Yet, the use of trypsin is considered to be a harsh method of detaching cells. Many researchers claim AccutaseTM to be a “softer” option of cell detachment when compared to trypsin.
Trypsin is a serine protease of animal origin. It breaks down the anchorage proteins that attach the cell to the culture vessel. After roughly 5 minutes, the cells will appear rounded and are now in suspension.
To complete the process of passaging, the effects of trypsin have to be inhibited by serum or other inhibiting molecules, such as soybean trypsin inhibitor. It is advisable to rapidly wash the cells and dilute the solution after detachment – a step that is not necessary when using Accutase™.
Trypsin introduces various unwanted side effects. After trypsinization, large quantities of glycopeptides, sulfated glycosaminoglycans, and hyaluronic acid can be found outside the cell. Also, trypsin leads to the release of a large portion of the cells’ total sialic acid.
Exposing cells to trypsin for a prolonged time will damage them. The surface will suffer damage and internally the cell will be harmed, e.g., degradation of polyribosomes.
Usually the cell will recover, but keep in mind that you want your cells to be in a stress-free state to gain reliable experimental data that is not altered by the side effects of trypsin.
Accutase™ serves as a direct replacement for trypsin and collagenase applications:
The efficiency is way higher than mammalian trypsin and collagenase, therefore Accutase™ can be used at a much lower concentration, making it less damaging to the cells.
Further, Accutase™ is:
Apart from cell detachment, Accutase™ can also be used to dissociate cell clumps. If the dissociation with Accutase™ is not successful, we recommend that you try Accumax™, which contains the same proteolytic enzymes but at a higher concentration.
about their experience with Accutase™
I like that Accutase is gentle on cells and auto-inhibits at +37 °C without the need for a neutralizing solution like with trypsin. Also, the cost of Accutase is less than trypsin + trypsin neutralizing solution, so it is cost-effective. It is also simpler to use, because it doesn’t require the extra neutralizing step, I just lift my cells in Accutase, then add them directly into fresh media.
I believe everyone in my lab now uses Accutase. We work with a wide range of cancer cells and neuronal cells too, and it works great.”
“We regularly use Accutase in cell culture and FACS applications using human embryonic stem cells and human induced pluripotent stem cells because these products give the best cell dissociation without compromising the health of our cells.
|Appearance||Clear, pale red to orange solution (anything in between)|
|Storage and shelf life|| (long term, ≤ -15 °C)
(2 months after thawing, +2 to +8 °C)
Do not store at room temperature.
|Shipping conditions||(dry ice)|
|Working concentration||1X (ready-to-use)|
|Accutase enzymes||1 x|
Accutase™ is a ready-to-use product, and you do not need to further dilute it before use. Do not pre-warm to +37 °C!
We advise you to not be stingy when using Accutase™ and use enough product, as opposed to detaching the cells mechanically (by pipetting up and down) – this can damage and kill your cells!
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.
Accumax™ contains the same enzymes as Accutase™ (proteolytic and collagenolytic) but at a higher concentration (3-fold). Further, Accumax™ comes without phenol red.
In general, you can use Accutase™ for all of your trypsin applications. Should you experience difficulties in dissociating cell clumps, you can try Accumax™ instead, which usually does the trick.
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!