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TRYPSIN EDTA SOLUTION
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INSTRUCTIONS:
TO REMOVE CELLS FROM CULTURES ATTACHED TO THE BOTTLE:
1. Remove by inversion or aspiration all the medium inside the bottle.
2. Add about 0.1 ml/cm2 of the Trypsin/EDTA solution in order to rinse the cell sheet and remove the remnant serum. The culture medium or the balanced solution can be used in large amount, without serum, which allows saving more of the trypsin solution.
3. Add about 0.05 ml/cm2 of Trypsin/EDTA solution under cell surface and look through microscope. When the cells acquire a round and individualized appearance, with dislodged from the tube, add 0.2 ml/cm2 of the medium with 10% serum. Serum has alfa 1-anti-trypsin which helps to neutralize the action of trypsin. If monolayer, the detachment can be seen with the naked eyes, and the medium will be presented as it has thin sand. It is important to first detach the cellular layer to then neutralize with serum, once that the lipoprotein membrane suffers a proteolytic action of the trypsin.
4. Then proceed with counting for dilution, freezing, etc.
TO REMOVE CELLS FROM ORGANS OR TISSUES:
1. Wash well the organ or tissue in medium without serum.
2. Using an Iris scissor cut the organ in small pieces (+1mm).
3. Aspirate all the supernatant
4. Add Trypsin/EDTA 3 to 10 times the correspondence volume for cut tissue.
5. Mix gently in an Erlenmayer, using a sterile magnetic agitator covered with PTFE. The collection of trypsin + cells can have intervals of 10, 20 or 30 minutes.
6. Elapsed the time for trypsin, aspirate the supernatant and transfer into tubes with complete medium + 10% serum. For each 10 ml of supernatant add one ml of complete medium with serum. The extraction of cells from the Erlenmayer can continue if you add more Trypsin/EDTA.
7. Centrifuge for 10 minutes at 1000 RPM the tubes with trypsin + complete medium, discard the supernatant and add a new medium.
8. Mix well and distribute the cells into culture flasks according to cell vitality and density.
PRESENTATION: Bottles with 75 ml --w/ trypsin (2.5g/L) with activity 1:250
SHELF LIFE: 12 months
STORAGE: 0 to -20°C
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DEFINITION: It is a balanced solution with trypsin, without calcium and magnesium ions and with a chelating agent.
ACTION: It is used to dislodge cells from culture bottles and/or for disaggregation of cells due to its enzymatic activity upon intercellular proteins. It also changes the membrane stability by removing calcium ion.
PRODUCT SPECIFICATIONS: It is used for the dissociation of cells from organs or tissues, even cultured in mono or plurilayers forms.
ADVANTAGES: As the process of dissociation and disaggregation of cells can be seen at microscope, it is possible to interrupt the process by adding a medium containing human or animal serum.
PHYSICAL-CHEMICAL CHARACTERISTICS:
1.) Appearance: clear rosy solution
2.) pH : 7.0 + 0.2
3.) Osmolality: 295 + 5%
STORAGE: After thawing, use the serum promptly and refreeze for future use. As it is a proteolytic enzyme, it may suffer a self-digestion which decreases its activity. Alterations for values close to 7.9 (more rosy) and the use at 37°C causes an increase on its activity, having as consequence diminution of pH and temperature. |
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