Dear all,
I am differentiating BM cells into BMDM using L929 conditioned media. Briefly, at day 0, I isolate BM cells from mice and seed it to 100pi petri dish using complete with 30% LCM (BMDM media). At day 1, i collect non-adherent cells and seed it (1x10^6 per dish) to 100pi culture dish using BMDM media. And i add 10ml of fresh BMDM media at day 4 and change half of this media to fresh BMDM media at day 7.
In this process, my cells differentiate too much and reach 100% confluency at day 5~6. Non-differentiated cells floats or attached lightly above the monolayer of differentiated cells. I wash them with 1X PBS before detachment of BMDM to eliminate them, but is it okay to just run the experiments? I'm afraid that i may have done something wrong during culture.
Also, morphology of differentiated cells are different with other reference images. Mine is more attached to the dish than other images, and have morphologies close to round shape with several short branches like LPS-stimulated cells. It also has something looks like vacuoles. I attached images which is similar with my cells.
I want some clues for my questions.
Thank you for reading.
The images look like activated macrophages, which makes me suspect LPS/endotoxin is present in your L929 conditioned medium. You should test your conditioned medium for endotoxin activity.
The other potential cause of this phenotype is that 30% L929 conditioned medium is actually a significant amount of M-CSF, enough to cause overgrowth.
In colony assays that were done in Richard Shadduck's lab, 50 ul of 1/2x LCM would yield 150 colonies from 100,00 mouse BMC in 1 mL assay plates, which translates to 3000 Units/mL. 30% LCM would be 900 Units/mL. We always used 50 units per microgram as a specific activity for M-CSF; that would mean your cultures have on the order of 18 micrograms per mL M-CSF. You should try lower concentrations of your L929 conditioned medium, like 5% or 10%.
In addition, when I worked with Christa Mueller-Sieburg prior to coming to work with Dr. Shadduck, we were characterizing the growth factor for bone marrow stromal cells, which was later shown by Elena Deryugina to be M-CSF. The concentration that induces stromal cell growth and differentiation was equivalent to 20% L929 conditioned medium or 10 micrograms/mL M-CSF. So you may be generating a mixed culture of BMDM and BM stromal cells, which have a fibroblastic morphology.
Alternately, you can avoid the potential complications of working with conditioned medium by using commercially available mouse M-CSF.
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