Is there an upper limit for the amount of time cells can be incubated with TrypLE Express Enzyme, TrypLE Select Enzyme, or CTS TrypLE Select Enzyme for dissociation?
There is no upper limit for TrypLE enzyme incubation. You can incubate your cells with TrypLE longer to detach cells. 30-45 min at 37 degrees C will be usually fine. Or you can consider using TrypLE Select Enzyme (10X) (Cat. No. A1217701 or A1217702 or A1217703) or CTS TrypLE Select Enzyme (Cat. No. A4738001) depending upon the application.
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For your TrypLE products, how does the enzyme get inactivated?
Our TrypLE enzymes are very gentle, and there is no need to use serum or individual proteins to inactivate the enzyme. The TrypLE enzyme activity is inactivated by dilution using the following procedure:
After cell dissociation, transfer the cell suspension to a 15-mL conical tube. Centrifuge at 100 x g for 5-10 min to remove residual enzyme activity. Discard the supernatant and resuspend the cell pellet in 2-5 mL of pre-warmed complete medium.
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Why are my cells not adhering when I transfer them to a new flask?
Trypsinize this type of cells for a shorter duration or use less trypsin or Gibco TrypLE Select or Gibco TrypLE Express Trypsin Replacement. After trypsinization dilute in 2 to 5 mL of cell culture growth medium transfer the cell suspension to 15 mL centrifuge tube. Centrifuge for 5-10 minutes at 100 x g. Discard the supernatant and suspend cell pellet in 2-5 mL fresh medium. Determine the count and seed the flask according to normal protocol and cell should adhere to the surface.
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Do you have a non-animal-origin enzyme for cell dissociation?
Yes, the TrypLE products contain rProtease, a non-animal, trypsin-like enzyme used for the dissociation of attachment dependent cell lines. TrypLE enzyme has demonstrated the ability to dissociate cells cultured both in serum-free and serum-supplemented systems. The catalog numbers for TrypLE Select are 12563-011 (100 mL) and 12563-029 (500 mL). Some example catalog numbers for TrypLE Express include 12604-013 without Phenol red (100 mL) and 12605-010 with Phenol red (100 mL). Both formulations are also available in additional sizes. All these products are animal origin-free.
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What is the difference between TrypLE Express Enzyme, TrypLE Select Enzyme, and CTS TrypLE Select Enzyme?
All the above products contain rProtease, a non-animal, trypsin-like enzyme formulation used for the dissociation of attachment-dependent cell lines. This alternative for porcine trypsin is a recombinant enzyme derived from microbial fermentation.
The TrpLE Express, TrypLE Select, and CTS TrypLE Select products differ in their production and testing methods and in the documentation provided.
TrypLE Select is for the industrial market and has release-testing both for endotoxins and enzyme activity. This material also has a Drug Master File on record. It is manufactured in AOF-dedicated equipment and is intended for Research Use/Further Cell Culture Manufacturing.
TrypLE Express is manufactured without use of dedicated equipment and is intended for Research Use Only. Not for use in diagnostic procedures.
CTS TrypLE Select is manufactured under scalable cGMP conditions using dedicated equipment and is designed for clinical applications. This product has a Drug Master File on record and is intended for Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. Not intended for direct administration into humans or animals.
To read more about TrypLE and cell release times using this reagent, please go here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html#1).
Find additional tips, troubleshooting help, and resources within ourMammalian Cell Culture Basics Support Center.
