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T4049 Sigma

Trypsin-EDTA solution

0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red

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Description

Components

Trypsin consists of a single chain polypeptide of 223 amino acid residues, produced by the removal of the N-terminal hexapeptide from trypsinogen which is cleaved at the Lys - lle peptide bond. The sequence of amino acids is cross-linked by 6 disulfide bridges. This is the native form of trypsin, beta-trypsin. BETA-trypsin can be autolyzed, cleaving at the Lys - Ser residue, to produce alpha-trypsin. Trypsin is a member of the serine protease family.

Biochem/physiol Actions

Trypsin cleaves peptides on the C-terminal side of lysine and arginine residues. The rate of hydrolysis of this reaction is slowed if an acidic residue is on either side of the cleavage site and hydrolysis is stopped if a proline residue is on the carboxyl side of the cleavage site. The optimal pH for trypsin activity is 7-9. Trypsin can also act to cleave ester and amide linkages of synthetic derivatives of amino acids. EDTA is added to trypsin solutions as a chelating agent that neutralizes calcium and magnesium ions that obscure the peptide bonds on which trypsin acts. Removing these ions increases the enzymatic activity.

Serine protease inhibitors, including DFP, TLCK, APMSF, AEBSEF, and aprotinin, amongst others, will inhibit Trypsin.

Caution

This product is stored frozen between -10 and -40°C. Repeated cycles of freezing and thawing should be avoided.

Preparation Note

This product does contain phenol red. Due to shipment on dry ice, there could be significant carbon dioxide buildup in the package. This CO2 may enter the solution and lower the pH slightly, giving an orange rather than pinkish color. The orange solution will still be suitable for use, or the pH can be adjusted with sodium hydroxide. Incubating cells with too high a trypsin concentration for a long period can damage cell membranes and kill the cells. Solubilizing trypsin or diluting it from a concentrated solution should be done with a buffered salt solution containing no Ca2+ or Mg2+.

Application

The typical use for this product is in removing adherent cells from a culture surface. The concentration of trypsin necessary to dislodge cells from their substrate is dependent primarily on the cell type and the age of the culture.

Trypsin-EDTA solution was used:
• in detaching HT29 human colorectal cancer cells cultured in RPMI 1640 which was supplemented with 10 % fetal calf serum, during relative cell frequency determination of high concentration samples.
• to trypsinize the transient transfected human embryonic kidney tcA-201 cell line.
• to enzymatically release mouse fibroblasts cells (cell line L929) adhered to the scaffold, during cell culturing to assess the influence of several modified treatments of Poly(L/D)lactide 96/4 non-woven scaffolds and fibres.

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Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport
WGK Germany 
3
Protocols & Articles

Protocols

Protocol for the Cryopreservation of Cell Lines

This protocol is recommended by the European Collection of Cell Cultures (ECACC) for the cryopreservation of its cell lines. This protocol employs the use of passive methods involving an electric –80...

Removal of Adherent Cells from Culture Surface Using Trypsin

Trypsin may be used to remove adherent cells from a culture surface. Cells are most commonly removed from the culture substrate by treatment with trypsin or trypsin/EDTA solutions. Trypsin concentrat...

Subculture of Adherent Cell Lines

Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. At this point the cell lines should be sub cultured in order to preve...
Cook Book Sept 2010 Volume 12, Fundamental Techniques in Cell Culture Laboratory Handbook-2nd Edition
Keywords: Adhesion

Peer-Reviewed Papers
15

References

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