Accumax
Non-mammalian and non-bacterial based / Cell detachment solution
Accumax is a ready to use non-mammalian, non-bacterial replacement for all applications of trypsin and collagenase in tissue dissociation, cell counting, and dissolving cell clumps such as spheroids. Accumax contains the same enzymes as Accutase and is a direct replacement for collagenase, and can be used to increase the accuracy of a cell count. It can also be added to a clumpy sample on a cell sorter to extend the sort time of the sample.
Advantage of Accumax
- Dissociates tissue.
- Increases accuracy of manual or automated cell counts.
- Used to dissolve neuronal and prostate spheroids.
- Removes cells from 3-D matrixes.
- Removes cells from hollow fiber cell reactors.
- Extends the sort time of clumpy cell on a sorting flow cytometer.
>> Click here to Accutase™ Cell Dissociation Solution
Cell Lines tested
A few cell lines that Accumax has been shown to detach without harm:
| hESCs | fibroblasts |
| keratinocytes | vascular endothelial cells |
| vascular smooth muscle cells | hepatocytes |
| hepatocyte progenitors | primary chick embryo neuronal cells |
| bone marrow stem cells | adherent CHO cells |
| adherent BHK cells | macrophages |
| 293 cells | L929 cells |
| immortalized mouse | testicular germ cells |
| 3T3 | Vero |
| COS | HeLa |
| NT2 | MG63 |
| M24 and A375 metastatic melanoma | gliomas U251 and D54 |
| HT1080 fibrosarcoma cells | Sf9 insect cells |
Applications
Accumax performs exceptionally well for:
Accumax is used to digest primary tissue, create single cell suspensions for cell counts, and declump cells for magnetic or flow cytometry cell sorting and removing from artificial growing matrixes.
| hESC culturing | virus growth assay |
| analysis of cell surface markers | cell proliferation |
| apoptosis | cell haptotaxsis |
| tumor cell migration assays | routine cell passage |
| flow cytometry | quiescence assays by serum starvation |
| transformation assays by oncogenetransfection | transfectionneural crest cell migration assays |
| production scale-up (bioreactor) | neural crest cell migration assays |
Increase in Cell Counts
 |
Various constructs of genetically engineered CHO cells, BHK cells, and a hybridoma were grown in suspension in serum-free or protein-free medium. Representative cell aliquots were treated with an equal volume of PBS or Accumax and incubated for 5 minutes at 37C. Cell number was then determined with a Coulter Counter. |
Primary Cell Dissociation Protocol for Accumax
This protocol for using Accumax to dissociate cells from primary tissue is a general-purpose protocol and may not be applicable to all tissue types. The individual investigator needs to optimize the conditions for his/her tissue specimens. Keep in mind that Accumax is a powerful enzyme mixture that can potentially dissolve not only the connective tissue of solid tissue but some fragile cell types as well if not closely monitored.
Materials
Sterile:
Accumax (Should be defrosted overnight in the refrigerator or in a bucket of room temperature water - not a 37°C bath)
DPBS (calcium and magnesium free)
Culture medium, i.e., DMEM/F12 with 10 – 20% FBS (or other appropriate media)
Pipettes - 1 ml, 10 ml
Petri dishes -100 mm, non-tissue culture grade
T25 culture flasks
Centrifuge tubes, 15-50 ml, depending upon the amount of tissue being processed
Scalpels
Forceps
Non-sterile:
Platform rocker
Trypan Blue
Microscope
Centrifuge
Procedure:
- Transfer the tissue to a petri dish containing fresh, sterile DPBS, and rinse.
- Transfer the tissue to a second dish; dissect off unwanted tissue, such as fat or necrotic material.
- Using two crossed scalpels or a scalpel and forceps, cut the tissue into small pieces approximately 1 mm in size.
- Transfer the tissue pieces to a 15 or 50 ml sterile centrifuge tube containing fresh, sterile DPBS.
- Allow the pieces to settle and carefully remove the supernatant. Repeat this wash step two times.
- Transfer the tissue pieces to a fresh petri dish and add enough Accumax to the plate to cover tissue.
- Incubate the samples on a platform rocker at room temperature 5 to 60 minutes. The tissue will “smear” on the bottom of the dish when the disaggregation is effective. To release more cells, gently agitate the sample by pipetting several times. It is best to check cell viability several times during the incubation using Trypan blue.
- Once disaggregation is complete, transfer the cells to a sterile centrifuge tube and centrifuge at 300 x g to pellet the cells and to remove the cell debris if desired.
- Carefully remove the supernatant and re-suspend the cell pellet in 5 ml of DMEM/F12 containing 10 – 20% FBS (or other appropriate media). Seed in a T25 flask. Replace the media after 48 hours.
Alternatively:
If cell isolation is from a soft tissue (such as liver):
- Transfer the tissue to a petri dish containing fresh, sterile DPBS, and rinse.
- Transfer the tissue to a second dish; dissect off unwanted tissue, such as fat or necrotic material. Add 1 – 2 ml of Accumax and use forceps to gently “tease” the cells into the Accumax.
- Residual connective tissue may be separated by allowing the pieces to settle or by filtration, if desired.
- Centrifuge the sample at 300 x g to pellet the cells and to remove cell debris if desired.
- Carefully remove the supernatant and re-suspend the cell pellet in 5 ml of DMEM/F12 containing 10 – 20% FBS (or other appropriate media). Seed in a T25 flask. Replace the media after 48 hours.
Increasing Reproducibility of Cell Counting with the Use of Accumax
Regardless of what the manufacturers of cell counters tell you, when counting cells with either a manual or automated method, the accuracy and the reproducibily of the counts will be increased if the cells are not clumped together. Accumax solution can be used to dissociate clumpy cells that are being counted. Accumax is gentle enough that an aliquot of it can be added to an aliquot of cells and allowed to set for a period of time without damaging the cells.
- Harvest a representative sample of clumped cells to be counted, 0.5 or 1.0 ml, and place in a 12x75 mm tube.
- Add an equal volume of Accumax to the sample of cells, and incubate for 5 to 10 minutes at room temperature.
- Count the cells by your normal procedure. Note that the cells have been diluted an extra 2 fold.
Q&A
A: Accutase can be used whenever gentle and efficient dissociation of any adherent cell line is needed. Accutase is a direct replacement for trypsin.
Q: What is the difference between Accutase and Accumax?
A: Accumax contains the same proteolytic and collagenolytic enzymes as Accutase, but is formulated at a concentration that is 3X higher and does not contain phenol red.
Q: What color should my Accutase be when it arrives?
A: Please see this document for information, including photos, about the effects of shipping on the color of Accutase - what's normal and what's not.
Q: Can I use Accutase to dissociate non-adherent clumpy cells?
A: Yes. Accutase can be used to dissociate spheres of neural progenitors (neurospheres). However, if the clumps do not dissociate completely, then consider using Accumax which contains the Accutase enzymes, but at a higher concentration.
Q: The frozen bottle of Accutase shows uneven color distribution and upon thawing it has layered color distribution. Is this normal?
A: Yes. During the defrosting procedure or shipping, uneven color can be observed. However, this will not compromise the activity or performance of Accutase. Just be sure to mix the defrosted Accutase by inverting the bottle to get even color before use.
Q: The Accutase or Accumax arrived partially thawed. Can I still use it?
A: Yes. As long as the product contains an "ice-cube" or is cool to the touch it can be placed in the refrigerator for later use. It also can be refrozen for long term storage (if frozen within two months of receipt).
A: If it was exposed to 37 °C just until complete thawing was achieved without raising its temperature to 37 °C, it still can be used. However, it could result in decreased enzyme activity and it may take more time to get full dissociation. If you observe this, consider starting with a new bottle. If a bottle of Accutase or Accumax is kept at 37 °C for more than one hour, it will lose its activity.
Q: Do I need to aliquot and refreeze Accutase or Accumax after I defrost a bottle?
A: No. Once thawed, it is stable for at least 2 months in the refrigerator if stored promptly after use.
Q: Do I need to wash my cells before adding Accutase?
A: No - pour off the media and add enough Accutase to cover your cells and wait for them to "ball up".
Q: Do I need to stop the dissociation action of Accutase with serum?
A: Usually not. Accutase is gentle enough that only dilution of the reagent with DPBS or media is required to stop the dissociation activity. In the unusual cases where inactivation is required, the standard trypsin inhibitors will work, such as soybean trypsin inhibitor.
Q: Do I need to wash Accutase out after using?
A: No, this is one of the advantages of using Accutase.
Q: Do I need to dilute Accutase before use?
A: No. Accutase is supplied as a convenient, ready to use reagent. No dilution is required prior to use.
Q: How long should I leave the Accutase on my cells?
A: Detachment times for Accutase are similar to trypsin. This time should be determined empirically. Look under the microscope and watch them turn from spidery to little balls. In general, cells can be left in Accutase without damage for a much longer time than trypsin.
Q: What is Accutase made from?
A: Accutase contains no mammalian or bacterial components. It is a natural enzyme mixture with proteolytic and collagenolytic enzyme activity. This means it mimics the action of trypsin and collagenase at the same time. However, because it is more efficient than mammalian trypsin & collagenase, it is formulated at a much lower concentration making it less toxic, and gentler but just as effective.
Q: Do I need to worry about over-dissociating my cells with Accutase?
A: No. However, although Accutase is gentle on cells, the optimal time for dissociation should be determined for your specific cell type and application.
Reference
Downloads
- Primary Cell Dissociation Protocol for Accumax
- Increasing Reproducibility of Cell Counting with the Use of Accumax
- Defrosting Accutase™ and Accumax™ Correctly
Ordering Information
| Product | Storage | Cat.No. | PKG Size | Price | |
|---|---|---|---|---|---|
| Accumax | -20°C | NU1708754 | 100 ml | 72.00 | Buy |