Nacalai USA - Innovations for Life Sciences

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Accutase™

non-mammalian and non-bacterial based

Accutase™ is a ready to use non-mammalian, non-bacterial replacement for all applications of trypsin. It is a natural enzyme mixture with proteolytic and collangenolitic activities. It mimics the action of trypsin and collagenase simultaneously. However, because the enzymes operate more efficiently than trypsin and collagenase, it results in gentler and more effective cell detachment.

Advantage of Accutase™

  • Gentle and efficient dissociation of any adherent cell line
  • No mammalian or bacterial components are contained
  • No neutralization steps by serum or trypsin inhibitors are required
  • Works extremely well on embryonic and neuronal stem cells

Cell Lines tested

A few cell lines that Accutase™ 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

Cell Detachment Result

Human MG63 Fibrosarcoma cells cultured in DMEM + 10% FBS were treated with Accutase™. Treatment resulted in rapid cell detachment, a single cell suspension, and high viability. Accutase is gentle on cells; viability was 97 ± 3% even after 45 minutes in Accutase.

Applications

Accutase performs exceptionally well in detaching cells for:

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 oncogene transfectionneural crest cell migration assays
production scale-up (bioreactor)  

Typical cell passaging protocol using Accutase™

Accutase is formulated at a concentration that is ready to use, once defrosted. (Note: Never defrost a bottle of Accutase at 37°C.) A defrosted bottle of Accutase can be removed from the refrigerator and immediately applied to cells. It does not need to be and should not be pre-warmed to 37°C. Accutase contains proteolytic and collagenolytic enzymes to gently break down the cell adhesion structure on the outside of cells that attaches them to the bottom of the flask.

This entire procedure should be done in a laminar flow hood using proper aseptic technique.

  1. Carefully aspirate all of the media from the cell culture flask. (Rinsing with PBS is not necessary.)
  2. Immediately add enough Accutase to the flask to cover the cells. (Typically 2.5 to 5ml for a T25 flask depending upon confluency and density of the cell culture.)
  3. Set the flask aside at room temperature (RT) for 5 to 10 minutes up to a maximum of 1 hr. Check the flask frequently to see if the cells have rounded rather than merely shrunken or no longer appear "spidery" while remaining attached to the bottom of the flask.
  4. Once the cells have turned into "balls", smack the flask against the palm of your hand to dislodge any "stickers".
  5. Gently disperse the cells and take a sample of the cell suspension to determine the viable cell density.
  6. Add an aliquot of the detached cells to fresh media in new flasks. Place the flasks into the 37°C incubator. No neutralization steps are required. The cells will reattach within a few minutes depending upon cell type.

Ordering Information

Product Cat.No. Storage Quantity Price(US$)  
Accutase™ NU1267954 -20°C 100 ml 20.00 Buy