Murine BMDCs - (Oct/15/2005 )
I´ve been culturing BMDCs according to Lutz et al (1999 - an advanced culture method for generating large quantities of BMDCs) and, after many dificulties, it seems to be working out now. However, this protocol takes too much time and care, since 12th day cultured cells mature spontaneously. I noticed that many other researchers are diferentiating dendritic cells only 6 days and would like to know if anyone is having good results with this experience. Aren´t the dendritic cell levels in culture to low?
Here´s a brief resume of the protocol that I´m currently using:
1. Extract BM of mice (centrifuge, etc - do not lise RBC)
2. culture DCs - DMEM FBS 10% and GM-CSF - for 12 days
3. on 12th day stimulates with maturation factors (such as LPS)
4. Assay maturation by Flow cytometry (CD11c, CD40, CD86, Iab).
Thanks for helping and good luck with you cultures!
Here's my protocol: (it's not much faster...I would also be interested in a 6 day culture!)
day 1 extract murine bone marrow, lyse RBC
and culture in 10% serum + GM-CSF
day 4 add fresh media
day 8 remove 10mL media (gets rid of most unwanted floating cells);
add 10mL new media (this has less GM-CSF)
STIMULATE with LPS (or another stimulation factor)
day 9 add more media
day 10 harvest DCs for further use (mainly want mature floating or loosely attached cells)
This past harvest I started with ~32 million BMCs and ended up with 104 million Mo-DCs