T4049

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

• Synonym(s): Cocoonase, Tryptar, Tryptase
• Enzyme Commission number: 3.4.21.4 (BRENDA, IUBMB)
• MDL number: MFCD00130286
• UNSPSC Code: 12352204
• NACRES: NA.75

Properties

• biological source: Porcine
• Quality Level: 400
• sterility: sterile-filtered
• product line: BioReagent
• form: solution
• mol wt: 23.4 kDa
• concentration: 0.25%
• technique(s): cell culture | mammalian: suitable; single cell analysis: suitable
• impurities: Porcine parvovirus, none detected (9 CFR)
• pH: 7.0-7.6
• shipped in: dry ice
• storage temp.: −20°C

Description

General description

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.

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.
• to dissociate cells from the culture dish for flow cytometry analysis.

Suitable for use in the preparation of single cell suspension for sequencing.

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.

Components

Trypsin Solution (2.5 g/l porcine trypsin and 0.2 g/l EDTA•4Na in Hank′s Balanced Salt Solution with phenol red, 1X, cell culture tested)

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⁺.

Safety Information

• Storage Class Code: 12 - Non Combustible Liquids
• WGK: WGK 1
• Flash Point(F): Not applicable
• Flash Point(C): Not applicable