Non-mammalian and non-bacterial based / Cell detachment solution
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.
>> Click here to Accumax™ Cell Dissociation Solution
A few cell lines that Accutase™ has been shown to detach without harm:
|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|
|M24 and A375 metastatic melanoma||gliomas U251 and D54|
|HT1080 fibrosarcoma cells||Sf9 insect cells|
Accutase performs exceptionally well in detaching cells for:
|hESC culturing||virus growth assay|
|analysis of cell surface markers||cell proliferation|
|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)|
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.
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.
Primary macrophage cultures may be derived from density gradient separated whole blood or bone marrow by culturing these isolates in the presence of specific growth factors in order to encourage the growth, expansion and differentiation of the macrophage cell type. A literature search will yield several in-depth protocols which out-line these isolation techniques in detail. Once harvested and put into culture at 37oC in a 5% CO2 incubator the macrophage progenitor cells will adhere to tissue culture flasks and will not be washed away when the media is changed in order to remove any non-adherent cells and allow for macrophage differentiation/maturation.
To remove the Primary macrophage cultured cells from the tissue culture plates for further analysis:
Passaging Macrophage Cell Lines such as DH82 and RAW264.7
Using Accutase™ Cell Detachment Solution
For those investigators who prefer to utilize immortalized cell lines rather than isolate primary cultures, Accutase™ may be used to passage their macrophage lines in a similar manner to the techniques used for most other adherent cell lines.
Please note that different adherent cells stick to tissue culture plastic with varying degrees of adherence. For this reason the incubation time required for detachment can vary with some cell types needing more or less time to detach. In the case of extremely tenaciously adherent cells our stronger formulation, Accumax may work better.
Dissociation of adherent human or rat NSCs
Dissociation of human or rat neurosphere cultures