Hi all,
I isolated murine BMDM. I trypsinized them after 10 days in L9299 medium and seeded them in DMEM + glutamax, mem and 10% FCS. However after passaging my cultures are not very healthy. Do I need to keep them in L929 and what is the best concentration for seeding the cells at this point.
Thanks!
The DMEM medium you use should be supplemented with 15% of conditioned medium derived from L-(29 cultures along with 10% FBS and antibiotics. The isolated bone marrow cells should be placed in this medium in NON-tissue culture treated petri dishes and incubated for 3-4 days before the first medium change. At the first medium change, non-adherent cell should be rinsed and removed. To passage macrophage cells, I find it better to use gentle mechanical scraping with a flat spatula-type scraper. Trypsin does not work well to detach macrophage and the length of time it takes to work damages the cells and creates low viability. I do not use macrophage beyond passage 2. You can grow them up in the petri dishes, scrape collect and centrifuge them in preparation for cryostorage. They tend to terminally differentiation the longer they are kept in culture. Only when I am ready for experimentation and terminal differentiation do I place the cells in tissue-culture treated flasks.
Hi Astrid,
I am sharing my protocol for isolation and maintenance of murine BMDMs.
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