Hello everyone,
I came across a question while harvesting my U2OS transfected cells.
After trypsination, I resuspend my cells in cold medium with 10% FCS and I centrifuge them into four different pellets for downstream RNA/DNA/PROT extractions and Cytometry study.
For the sake of saving some really expensive FCS, I was wondering if harvesting cells with just medium could be ok ?
It's important to note that
1- no cells are put back into culture
2- cells only stay in this cold medium for 10 minutes max before being pelleted, removal of supernatant and freezing at -70°C.
We all well know that medium+FCS will inactivate trypsin but in this case those it really matter ?
Thanks for your insight !
Philippe.
Hello Philippe Paget-Bailly
Yes, it does matter. You need to inactivate trypsin before you use the cells for downstream applications, especially protein extraction and flow cytometric study.
Long-term incubation with trypsin will damage cells by striping cell surface proteins. Due to the proteolytic activity of trypsin, not only membrane proteins of the cell may be damaged but also trypsinization has been shown to down-regulate growth- and metabolism-related protein expression and up-regulate apoptosis-related protein expression. These may affect your results, especially when you carry out flow cytometry and protein based studies.
If you want to save FCS since it is expensive, I recommend the use of non-enzymatic cell dissociation solution which is composed of a proprietary mixture of chelators gently dislodging adherent cells and representing an optimized alternative to protein-digesting enzymes. Cells can be exposed to this solution for longer periods of time without the risk of damage associated with protein digestive enzymes like trypsin.
Best.
Typically, we use cold PBS for washing post-medium removal. Instead of trypsinization, we add the appropriate lysis buffer/Trizol and utilize a cell scrapper to detach the cells.
Vipin Bhardwaj Thanks for your answer !
I would also go that way if I could but with 20 conditions to harvest at the same time we went with F25 culture vessel that we harvest and pellet.
It saves us the hassle of having to scrap cells for downstrem protein extraction while having to fixate or cells for cytometry, plus pellting cells for DNA exptraction, plus rNA extraction...........
It is also easier and less time consuming to transfect/change medium of 20 T25 flask rather than 20 * 4 wells of 6-well plates. The downside is obviously the use of trypsin which, as Malcolm Nobre pointed, will damage your study material.
"When you pray for the rain, you have to deal with the mud" ^^
I'm curious to know why would you scrap cells in Trizol, the phenol does not completely destroy yours cells ?
To conclude, Leonid Ushanov thanks for the straight answer héhé! Like I said our cells only spend 10 minute in ice cold medium with trypsin so I was not that worried. Still, I had to ask.
Anyway, thanks for your time !
Philippe.
As long as all samples are treated the same way and you work fast, you are fine.
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