Hello,
I am new to harvesting BMDC and my most recent experiment had 45% CD11c+ cells that were viable (also many dead cells, only 20-30% viable).
I followed the method from Madaan A, et al. 2014. J Biol Methods - stepwise procedure for isolation of murine BMDC. Briefly, plated 10 million cells isolated from mouse femur into petri dish. Grew for 6-8 days with 10 ng/ml of GM-CSF and IL4.
Most papers seem to have 80% or more viable CD11c+ cells with this method. Are there any tricks? I seem to be missing something.
Thank you,
The protocol is pretty standard and it should give you a high number of DCs.
During the first media change, when you remove the media, only take out about 2/3. Keep the plate flat on, do not tilt it. Also aspirate with from the top of the media, not the bottom. This allows you to get rid of as many dead cells as possible without disturbing the live cells which usually sink lower.
I don't use RPMI (McCoy's 5A medium instead), but I really don't think that this will make too much difference.
Something else you can do at the end is once you isolated the non-adherent cells after differentiation, do a positive selection for Cd11c, it'll give you a pure population of DCs to start.
Good luck.
Thank you!
I normally do not remove the media - I just add fresh media with GM-CSF/IL4 every other day. Perhaps I should remove media as mentioned above to get rid of more dead cells?
Yes, you have to take some of the old media out to remove the dead cells. Living dead cells in the culture starts a lot of activation pathways that usually leads to more dead cells.