Does trypsin strip flask coating?

Does trypsin strip flask coating?

We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Mammalian cell/tissue cultures sometimes require flasks coated with proteins. My uneducated guess is that these proteins mimic the ECM, perhaps the basal lamina, so finicky contact-dependent cells can attach to it.

For instance, I have a BEAS-2B tracheal culture which requires a coating of albumin, fibronectin and type I collagen. This is applied by incubating an empty plastic dish or flask with a solution of these proteins overnight. I give this as an example of what a "coat" constitutes. You answer should be general enough to apply to at least the most common coat types, or if it depends on the coat, you should explain how.

Likewise, there is some variation in how the trypsinization occurs. To have a starting point, let's say I am trypsinizing according to typical ATCC recommendations: 5 minutes with 0.25% Trypsin-0.53 mM EDTA, then inactivate with equal volume medium. I haven't worked with a cell that can withstand this noticeably (but then I haven't worked with any exotic cells).

My question: When I trypsinize the cells to passage, what happens to the coat?

Since trypsin is a protease that cleaves even the attachment of cells to plastic, I am guessing that it will also destroy the coat. After the trypsin and cells are removed, the flask will have a tiny fraction of the protein coat that it had before, and will not support cells that require a coat any longer.

Am I correct in thus deducing that coated flasks cannot be reused after trypsinization? Are any of the alternative detachment reagents an exception to this?

Generally the coating is of poly D-Lysine. Proteases act only on L-amino acids. So, I guess nothing should happen to the usual coated flasks. I have reused the flask even after 2 rounds of trypsinization, and the cells seemed to be fine.

In your case, the matrix is made of proteins isolated from animal sources. They are susceptible to proteolysis. You are right to assume that these flasks should not be reused. If you have a shortage of flasks then you can buy uncoated petriplates (which are cheaper) and coat them yourself.

You should check the composition of the coating by using trypsin, e.g. poly-L-lysine or poly-D-lysine are usually applied:

Polymers of both D- and L-lysine are used to coat solid surfaces. Poly-L-lysine has been reported to improve the protein coating of ELISA plates. 6,7 However, in culture applications, certain cells can digest poly-L-lysine. In this situation, poly-D-lysine should be used as the attachment factor so that the cells are not disrupted by excessive uptake of L - lysine. The lower molecular weight poly-D-lysine (30,000 - 70,000) is easier to use because it is less viscous in solution, but the higher molecular weight poly-lysine (>300,000) provides more attachment sites per molecul e. The molecular weight poly-D-lysine often preferred by users is the 70,000 - 150,000.

  • Poly-D-lysine Hydrobromide - Sigma

Trypsin does not seem like to hydrolise poly-D-lysine. Roughly it is specific to L-Lys and L-Arg, so if your coating contains these amino acids (in your case it does), than it will be degraded.

Trypsin (EC3.4.21.4) is part of the serine protease family. Trypsin cleaves lysine and arginine at the C-terminal side of the peptide. The hydrolysis rate is slower if an acidic residue is on either sides of the cleavage site and no cleavage occurs if a proline residue is on the carboxyl side of the cleavage site. Trypsin optimum pH is pH-7 to 9. Trypsin will also hydrolyze ester and amide linkages of synthetic derivatives of amino acids such as: benzoyl L-arginine ethyl ester (BAEE), p-toluenesulfonyl- L-arginine methyl ester (TAME), tosyl-L-arginine methyl ester, N-α-benzoyl-L-arginine p-nitroanilide (BAPNA), L-lysyl-p-nitroanilide, and benzoyl-L-tyrosine ethyl ester (BTEE). Serine protease inhibitors that inhibit recombinant trypsin include TLCK (N-p-tosyl-L-lysine chloromethyl ketone), PMSF (phenylmethanesulfonyl fluoride), benzamidine, soybean trypsin inhibitor, and ovomucoid.

  • Trypsin Human, Yeast - ProSpec, or Sigma here

After incubation with 1mg/mL trypsin solution at 37°C for 1h, neither PLL nor PDL was found to be hydrolyzed, as indicated by the constant number of molecules found on the surface. The reason is that the entire polylysine chain was adsorbed to the surface, and steric hindrance and electrostatic repulsion prevented trypsin molecules from approaching the cleavange sites. To make polylysine exposed and available to trypsin digestion, a DSU SAM surface was created. A uniform coating of 5nm chromium and 30nm gold was first introduced on the slides. On top of that metal coating, an amine-active cross-linker, DSU SAM, was formed to covalently immobilize AF532-PLL/PDL molecules. After the free peptide molecules were removed, the slide was incubated with 1mg/mL trypsin at 37°C for 1h. The trypsin and cut polylysine fragments were then thorougly washed off. Intact PLL and PDL molecules had similar fluorescence intensities and surface number densities before trypsin was introduced. However, it is clear that trypsin digested PLL but not PDL. From over 30 images of different areas of the slides, for PLL the average numbers of molecules counted were 227±16 and 0 per frame before and after hydrolysis, respectively. In contrast, the corresponding numbers for PDL were 241±15 and 234±20 per frame, respectively. As explained in Emil Fisher's analogy of the lock and key, the L-isomer can fit the active site of trypsin bettern than the D-isomer. One reason is that only the L-isomer undergoes a coil to α-helix transition upon binding with the enzyme, while the D-isomer remains disordered.

  • 2008 - Applications of single-molecule detection in early disease diagnosis and enzymatic reaction study

So the result depends on the type of the coating. Btw. I think the easiest way to decide would be to check it with an experiment. (Note that I know nothing about coatings, just trypsin.)

In case someone finds this thread and wants an answer. In this paper (J Biomed Mater Res A. 2005 Oct 1;75(1):1-13.) the authors show that trypsinization does indeed remove the protein coating from the culture vessel.


  1. Noel

    Do not take in the head!

  2. Victorio

    Never mind!

  3. Arridano

    You have understood not at all well.

  4. Wardley

    I apologize, there is a suggestion to take a different route.

  5. Jackson

    And the gas conflict is not over, and here you are all about your rub

Write a message