I isolate bone marrow from 10-14 wk old B6j wildtype mice. BM is differentiated for 7 days in DMEM supplemented w/ 20% L-929 medium, 10% FBS and 1% HEPES. On day 7 medium is changed for same medium as above w/o L-929 medium. I note on day 8 cells begin to change morphology (become rounded) and lift (supposedly die?). On day 9 cells continue to change morphology and lift (see attached). Literature review suggests BMDMs are stable post differentiation w/o L-929 medium. Does anyone have any insight or suggestions please? Please do not hesitate to ask for more information.
Do you feed your cells (add more conditioned medium) on day 4?
I don't like to use the cells after a long time (9 days). I normally differentiate my cells for 7 days. I stimulate my cells on day 7 and my experiment is completed on day 8 or 9.
Yes I do. I culture cells from one mouse in ten 58 cm2 tissue culture dishes (10 mL medium per dish). On day three I add 3 mL more conditioned medium. I think what you do is the most common practice hence the effects of the removal of L-929 medium may be missed? My experimental time points include 48 hrs (a dish unstimulated w/o L-929 medium is one of my controls). This is how I saw this effect. Thank you for your reply Andre.
Liberty
You only add 3mL medium? I add another 10mL of R20/30 (20mL total in the end). The protocol I use is the same used by the Zamboni group (check the article bellow). After the 7 day differentiation I perform my experiment in R10/5 medium. I have noticed that if I don't use this my cells get a little wierd.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015263
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