Trypsin-EDTA(1X) 0.05%

Trypsin is a serine protease derived from porcine pancreas. It is a single chain polypeptide of 223 amino acid residue with substrate specificity based on positively charged Lysine and Arginine side chains. Trypsin predominantly cleaves peptide chains at the carboxyl sides of Lysine and Arginine, except when either is followed by Proline. It is most commonly used for dissociation and disaggregation of adherent cells. Trypsin can also act to cleave ester and amide linkages of synthetic derivatives of amino acids. Also, ethylenediaminetetraacetic acid (EDTA), a chelating agent is often added to enhance enzymatic activity of trypsin solution. EDTA acts by neutralizing calcium and magne-sium ions that enhance cell to cell adhesion. The typical use for this product is in removing adherent cells from culture surfaces

Specification

This product is 0.05% Trypsin and EDTA in HBSS. Also, it contains sodium bicarbonate and phenol red in HBSS

Notes

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

The concentration of trypsin necessary to dislodge cells from their substrate is dependent primarily on the cell type, the age of the culture and confluency of cell cultures

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

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+.

Trypsin solutions should be clear of particulates and flocculent material. Do not use if the solution is cloudy or contains a precipitate. Other evidence of deterioration may include degradation of physical or performance characteristics

How to Us

Before starting the implementation of any protocol, proper concentration of Trypsin-EDTA0.05% (BI-1601) solution and duration of incubation used for dissociation should be determined empirically for individual cell lines. Generally, Time required for dissociation of cells from a surface depends on cell type, cell density, the potency of trypsin, serum concentration in growth medium and time since the last subculture

Remove the spent medium from the culture vessel by aspiratio

Wash the monolayer by adding balanced salt solution without calcium and magnesium (PBS Minus: BI-1401) to the side of the flask opposite the cells

Rinse the cell sheet by moving the flask gently for 1 to 2 minutes and discard the wash solution

Add 0.05% Trypsin-EDTA (BI-1601) solution to the side of the flask opposite the cells. The volume should be sufficient enough to completely cover the monolayer of the cells

Move the flask gently to ensure that the dissociation solution covers the cell sheet

Incubate the flask at 37°C for 2 to 3 minutes. Monitor the process by observing the flask under an inverted microscope. When dissociation is complete, the cells will be in suspension and appear rounded. In addition to moving gently, flasks of cell lines that are characteristically difficult to remove from substratum may be tapped to expedite removae

Note: please note that the exact time needed to dissociate cells will vary according to the cell line. The dissociation pro cess should be monitored closely to avoid cell damage

Once the cell dissociation is complete add serum containing complete medium to the flask to inhibit the tryptic activity which may further damage the cells.

Disperse the cells into a single cell suspension by pipetting repeatedly

Count and subculture the cells