Does anyone have experience using PefaBloc with Caco-2 cells? We need the Pefabloc to stop enzyme activity in simulated in-vitro gastric digests where pancreatin & bile extracts are used. The cells are incubated with the digests for 1-4 hours, then washed, given fresh medium and incubated a further 23-20 hours before collected. Pefabloc is meant to be safe in cells up to 0.25mM concentration however, in order to stop the full enzyme activity you need to have 1 to 4mM in solution. Just wondering if anyone has any experience in this context?
To preempt the question, yes, we know we could heat the digest but we don't want to do this as it risks damaging other components too.
See this link , maybe useful
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613083/
Hi Darja, I have the same problem as you. Did you solve it or did you already test a safe concentration of Pefabloc?
Elisa Meneghetti, we ended up doing a couple tests with Pefabloc but it always impacted the cells at concentrations, which by our calculations, were needed to inactivate all the enzymes. What we did instead, if you have literally the exact issue as us with gastric digests, is that we gave the cells fresh medium 24hrs prior to treatment with the digests instead of directly before the treatments -- this gives them time to produce some inhibitors (its the same concept as if you don't wash them with PBS before trypsinizing then the trypsin doesn't really work). We also used slightly less of the digests on the cells (diluted in medium). By doing this the cells were able to protect themselves from any adverse effects from the digestive enzymes without needing heat to inactivate them. We were looking for a stress effect from a digested compound so we also ran several 'blank' digestion fluid controls to check if it caused any stress and didn't see any, and there was no impact on cell viability either (MTT)
Darja, I'm reading this today and would like to ask you a question. What about freezing the digested samples right after the digestion? I have the same issue now, and I've read in the Infogest protocol that you could do that to stop the enzyme activity, without having to use Pefabloc. I don't know if the enzymes would keep on working after thawing to use in the cell culture. I was planning to snap freeze the digested samples and use it on the cell culture after thawing.
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