I am working with murine macrophages extracted from the peritoneal cavity. They easily attach to my 24 multiwell plaque, but it has been a living hell to detach them alive. I have tried to incubate them with PBS EDTA 10mM, Versene or Versene EDTA 4mM. I have tried to make the incubation at 37ºC and also on ice, both with awful results.
They practically don't detach at all, and I end up collecting 10.000 or so from the 200.000 that I had initially seed. I have also tried to scrape them but they die, so the results obtained are not valid.
If someone could give me a protocol that actually works or some clues I would really appreciate that.
I am clueless and unable to proceed with my thesis, all the help will be more than welcomed!!!
Have you tried trypsin?
I tried once with Trypsin at 37ºC for 3 to 5 minutes, followed by inactivation with RPMI medium + 10% FBS and centrifugation, and it looks like it worked. Of course some of the cells died, but it wasn't much. Moreover, pay attention on what you are looking for. I suppose trypsin may cause morphological changes or activate some cells' pathways.
Hello Judit Pelegrin Perez
Please refer to the research article below. This may help you.
Article Culture and recovery of monocytes and cell lines from tissue...
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Macrophages can be separated from other cell types by their ability to readily attach and spread on glass or on plastic surfaces which are treated for optimal growth of cultured cells (tissue culture-treated plastic). To detach macrophages from these surfaces, techniques must be used which require prior preparation of special flasks or vessels, utilize expensive equipment, are time-consuming and almost uniformly require that the macrophages be exposed to various chemicals. We now report that macrophages can be enriched and recovered efficiently after attachment to disposable polystyrene bacteriologic petri dishes simply by gentle scraping with a rubber policeman. In this paper we compare this method to others currently in use in which resident peritoneal cells, peritoneal exudate cells or cells from bone marrow-derived cultures are detached from treated dishes using cold shock, chelating agents and lidocaine. In all studies, advantages were noted when cells were incubated in untreated dishes and detached by gentle scraping. In addition, untreated dishes supported the growth of adherent cell lines IC-21 and L929B and yielded large numbers of cells, with high viability, which were easily harvested.
Best.
Use Accutase (Life technologies). It is very mild and recovery is really good.