We are setting up fibroblast cultures in a new location and we had to change the mediums suppliers. At the trypsinization step trypsin did not work and we realized our hanks medium has Ca and Mg. Does anyone know if these components inhibit the enzyme?
thank you very much for all your help!
Dear Dr. Ana Lopez,
When cells adhere to the substratum, they produce integrin proteins that help in the attachment of the cells to the substratum as well as to one another. Important cations such Ca2+ and Mg2+ are essential for maintaining protein activity. These cations help cells to adhere and grow.
During cell detachment, trypsin/EDTA is used. Trypsin is a protease that digests the adhesion proteins which hold the cells to the substratum. EDTA is a chelator that chelate divalent cations which are cofactors for protein activity. By removing magnesium and calcium, cells lose contact with each other, there is loss of adherence and the cells round up in solution.
When you use Hanks medium with calcium and magnesium, trypsinization will not work because trypsin activity will be inhibited. So, whenever trypsinization process is carried out, one has to ensure that calcium and magnesium cations are reduced, to favor trypsin activity.
Regards,
Malcolm Nobre
Thank you very much for you quick response. We have noticed other differences in the media we recently bought compared to our previously used ones. For example:
- Our new Phenol Red DMEM doesn´t have sodium bicarbonate
- The new DMEM withoud phenol red has sodium pyruvate.
Do you think that could alter the cellular growth?
Thanks in advance!
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