In our experiment, monocyte-derived THP-1 macrophages was used for infection with pathogenic bacteria and after Transmission electron Microscope Image, the main problem is, how do we detach theses infected monocyte-derived THP-1 macrophages form 25 cm2 culture flasks?
Hi,
Have you tried 0.025% trypsin slution containing EDTA?
It is what I used for all the adherent cell lines I worked with.
Alexis
If you are concerned about enzymatic Action then you could try using EDTA alone. This usually helps detach cells very readily.
Macrophages stick very firmly to plastic. We used to use un-treated plastic petri dishes to culture them in the hopes that they wouldn't stick as much. Since we were doing flow cytometry on them, we did not want to degrade the surface proteins with a protease like trypsin, so instead we rinsed them with a solution containing EDTA (don't remember the concentration) and them left them in this solution in the refrigerator for one hour. Then we would gently scrape them off.
I will certainly echo the comments above with regards to using EDTA. If you want to avoid using trypsin, I would highly recommend a 5-10 min treatment (at 37C) with sterile PBS containing 0.1% EDTA. It should work fine.
Treatment with edta only is the safest. alone is the safest lalitha kabilan
It is quite hard to detach. Try with cold PBS, or EDTA. But I would advice to use non-treated flasks, that makes cells less adherent and thus easier to detach.
It depends on your assay limitations: for flow cytometry, you could use a cell scrapper, because it won´t interfere with surface molecules. For functional analysis, without interfering with surface proteins, you could let the flasks on ice for 30min, and wash/collect cells with ice-cold PBS. If you want to analyse gene expression, you can remove the cells by enzimatic digestion, cell scrapper, cold PBS. Hope this helps.
The Corning Ultra Low Attachment Surface is a covalently bound hydrogel layer that is hydrophilic and neutrally charged. Since proteins and other biomolecules passively adsorb to surfaces through hydrophobic and ionic interactions, this hydrogel surface naturally inhibits nonspecific immobilization via these forces, thus inhibiting subsequent cell attachment.
This Ultra Low Attachment Surface has been shown to successfully inhibit attachment of anchorage dependent MDCK, VERO, and C6 cells grown for a period of time equal to that necessary to obtain confluent cell growth on the control surface (standard tissue culture treated polystyrene). This surface has also been shown to prevent the attachment and activation of macrophages and neutrophils
Sorry, but THP-1 cells are not suspension cells? I have seen from ATCC datasheet that they grow in suspension, and after treatment with PMA they start to attach to the flask.
I am just starting to grow this cell line so I would like to know how to detach. I have also bought the cell dissociation solution 1x non enzymatic from sigma. Can I use it?
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