Product: Filter sterilized solution of 0.5% Trypsin form porcine pancreas in 10x Dulbecco´s PBS (pH 7.0-7.6) with EDTA, without Ca2+, without Mg2+, and without phenol red. Concentrated cell detachment solution for breaking down cell adhesion structures.
Appearance: Clear colourless solution.
Storage: -20ºC for up to 2 years.
Activity: > 5000 BAEE U/ml
Osmolality: 280-360 mOsm/kg
Parvovirus test : negative
Performance: Cell Detachment
Product: ready-to-use cell detachment solution for breaking down cell adhesion structures located on the outside of cells that attaches them to plasticware.
Appearance: Accutase® contains phenol red as a pH indicator. If frozen, it should be yellow and upon thawing, it becomes an orange liquid.
Storage: -20ºC. Once defrosted, store at +2ºC-8ºC for up to 2 months. If needed, one can freeze the solution again, promptly after using. Accutase® should be defrosted overnight in a refrigerator or placed in a cold water bath. Accutase® should NOT be defrosted in a warm water bath as the enzyme mixture is sensitive to temperatures above 37ºC and enzymes will be inactivated at 37ºC after 45 minutes. After defrosting, mix the solution well, as the components will not melt evenly (one can notice a colour gradient), and use promptly. Store the remaining solution in the refrigerator as indicated.
Accutase® is a ready?to?use non?mammalian, non?bacterial replacement for all applications of trypsin. Accutase® 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. Can be used whenever gentle and efficient detachment of any adherent cell line is needed.
Accutase® is a direct replacement for trypsin. Works extremely well on embryonic and neuronal stem cells; mono layers of stem cells can be grown after passaging with Accutase®. Preserves most epitopes for subsequent flow cytometry analysis. Does not need to be neutralized when passaging adherent cells. The addition of more media after the cells are split dilutes Accutase® so it is no longer able to detach cells. Does not need to be aliquoted. A bottle is stable in the refrigerator for 2 months
Accutase® performs exceptionally well in detaching cells for: hESC culturing, analysis of cell surface markers, virus growth assay, quiescence assays by serum starvation, transformation assays by oncogene transfection, neural crest cell migration assays, cell proliferation, apoptosis, cell haptotaxsis, tumor cell migration assays, routine cell passage, production scale?up (bioreactor), and flow cytometry.
Preparation of 1x solution from 10x concentrated solution
Note: The entire protocol should be carried out in a laminar flow hood, using proper aseptic techniques.
1. Pre-warm the 10x concentrated Trypsin/EDTA solution to 37ºC by placing in a water bath.
2. Once thawed, aseptically dilute 100ml of the 10x concentrated solution with 850ml of a sterile Ca2+- and Mg2+-free salt solution (e.g. Dulbecco´s PBS) and mix well.
3. If necessary, adjust pH to 7.2-7.8 with 1M HCl or 1M NaOH.
4. Adjust the volume to 1000ml with sterile Ca2+- and Mg2+-free salt solution (e.g. Dulbecco´s PBS) and mix well.
5. Dispense the solution into sterile container and store at -20ºC.
Typical trypsinization protocol
(depends on cell line and should be adapted conform)
Prior to start, pre-warm all solutions (Trypsin/EDTA (1x) and PBS (without Ca2+ and without Mg2+)) to 37ºC by placing in a water bath. The entire protocol should be carried out in a laminar flow hood, using proper aseptic techniques.
1. Carefully aspirate off all the culture media from the flask, without disturbing the cell layer or letting the cell layer dry. Proceed immediately with washing the cells with PBS (rinse with 5 ml for a T25 flask and with 10 ml for a T75 flask).
2. Immediately after, add sufficient Trypsin/EDTA (1x) solution to cover the cells. For a T25 flask, 0,5ml* should be enough. Incubate the flask at 37?C for 2-3 minutes in the cell culture incubator. Check cell morphology visually (microscope) to verify if cell have rounded, if not continue incubation. The solution in the flask will appear cloudy. Incubation time should be kept at a minimum and overexposure should be avoided, as Trypsin can cause cellular damage. The required time depends among others on cell type, culture age, cell density, and the serum concentration in the growth medium).
3. Once cells are rounded and detached, beat the flask against the palm of your hand to loosen any remaining attached cells
4. Wash out all the cells from the surface by pipetting the fresh complete cell culture medium (5ml) all over the surface. Gently disperse the cells to break cell clumps. Take a sample to determine the viable cell density, and add aliquots of detached cells to fresh culture media in new flasks.
*) When using more, e.g. 2-4ml, then in step 4, you must add more serum containing medium to inhibit trypsin after digestion has been completed, or neutralize excess of trypsin by adding trypsin inhibitor.