Trypsin-EDTA Solution, 1X

1. Bring ATCC^® Trypsin-EDTA solution to the appropriate temperature (see cell line product sheet). This may be 4°C, room temperature, or 37°C depending upon the cell type. You may also need to use a balanced salt solution [e.g., ATCC^® Dulbecco's Phosphate Buffered Saline (PBS) without Ca or Mg, Catalog number 30-2200] to rinse the cells. If so, bring this to the same temperature. Finally, bring fresh, complete cell culture media to the appropriate temperature for cell growth (e.g., 37°C).
2. Remove and discard the cell culture medium from the flask.
3. Depending upon the cell line, rinse the cell monolayer with either 5 mL of ATCC^® Trypsin-EDTA solution or ATCC^® Dulbecco's PBS (for more trypsin-sensitive cells) and remove.
4. Add 2 to 3 mL of ATCC^® Trypsin-EDTA solution and incubate at the appropriate temperature (4°C, room temperature, or 37°C). Continually check the progress of cell dissociation by microscopy. To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for them to detach.
5. Once the cells appear to be detached (5 to 15 minutes for most cell lines, they will appear rounded under the microscope), add 6 to 8 mL of complete growth medium to the cell suspension and with a pipette wash any remaining cells from the bottom of the flask. Check the cells with the microscope to be sure that most (> 95%) exist as single cells. If cell clusters are apparent, continue to disperse the cells with gentle pipetting (see Troubleshooting, below).
6. Add 12 to 15 mL of fresh cell culture media to a new flask and equilibrate this media to the appropriate pH and temperature. Collect the cell suspension, count and/or divide it, and dispense the cells into the newly prepared flask. Refer to the cell line product sheet for recommended subcultivation ratios.
7. For serum free or low serum media, remove the ATCC^® Trypsin-EDTA solution by gentle centrifugation (5 minutes at 125 x g) and resuspend the cells in fresh medium.

Troubleshooting

Cells are difficult to remove.

• The dissociating agent is too weak. Try incubating at higher temperatures.
• Inhibitors in the medium (e.g., serum) are inactivating the trypsin. Rinse the cell monolayer more thoroughly before incubating with ATCC® Trypsin-EDTA solution.
• Cells have been at confluent density for a too long and the cell-to-cell junctions are so tight that they are preventing the enzyme from reaching the substrate-cell interface. Subculture cells before they are 100% confluent.

Cells clump after dissociation.

• DNA has been released from lysed cells because the dissociation procedure was too harsh. Add a drop of sterile DNAse (1 mg/ml in water) to the cell suspension. In the future, treat the cells more gently during pipetting, shorten the incubation period, and/or decrease the incubation temperature.
• Cells are reaggregating before subculturing. Hold the cell suspension on ice if there will be a delay between removing cells from the flask and dispersing them into fresh cell culture medium.

Cells have difficulty reattaching.

• The dissociating enzymes may have stripped necessary attachment proteins from the cell surface. Treat the cells more gently, use less ATCC® Trypsin-EDTA solution, shorten the incubation time, and/or lower the incubation temperature.
• Not enough serum or attachment factors are in the medium (common with serum-free medium). Add attachment factors or use protein-coated plates (collagen, polylysine, gelatin, etc.).
• ATCC® Trypsin-EDTA solution was not inactivated by the cell culture medium (e.g., the serum). Add specific enzyme inhibitors or remove the ATCC® Trypsin-EDTA solution by gentle centrifugation (5 minutes at 125 × g) followed by a medium change.

The product is provided 'AS IS' and the viability of ATCC^® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

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