I've got some problems growing Bl/6 mouse BMDC's, and didn't know how much information you all may have on the subject. The reagents I use are GMCSF(20ng/mL), IL4(10ng/mL), and LPS(100ng/mL), all concentrations listed are final concentrations in cell cultures. Typically, I harvest bone marrow, break it up into monocytes, plate them in complete media+GMCSF in 24 well plates at 3*10^5 cell/mL, then at day 2 I wash off all non-adherent cells, and give fresh media with GMCSF+IL4 every 2 days until day 6 without removing any more cells (I pull off media, spin, and resuspend in fresh media before adding back to the plate). At day 6, I pull off all non-adherent cells and replate in fresh media+GMCSF+IL4+LPS or other maturation factors, then run flow on day 7(24 hours post LPS).
I've been defining my mature DC's as CD80/86 high and CD11c+. When I flow and gate on living cells, then CD11c+ cells, I always have two really nice populations of CD80/86 high and CD80/86 low. The problem I've faced is that this population distribution isn't always consistent, and I'm struggling getting my LPS to work, which from the literature, should NOT be difficult. While trying to remedy this, I decided to see if IL-4 was responsible for maturing my dendritic cells. Some papers suggest that it does so strongly, others show that it only has mild effects. In my last cultures, I had about 80-85% CD11c positive cells. All wells that were given IL4 were about 80% mature, 20% immature, and there was no effect from my 24 hour LPS stimulation. In my IL4 negative cultures, only about 15-20% of my cells were mature, and the rest were immature. Once again, LPS had no effect on these cultures.
My current ideas on the problem are:
a)my IL-4 is contaminated (it should have less than 1 EU per ug protein)
b)my LPS is not functioning (interestingly, in a previous culture, I saw a modest increase in mature cells vs immature cells in LPS stimulated cultures...about 10-15%, which is still sub-par)
c)my DC's are not what they seem (although cultures with high percentages of CD80/86 high cells are generally non-adherent and covered in dendrites)
I am completely open to ideas or suggestions. In my next culture I plan to add MHCII to my flow staining mix to verify that my CD11c+ cells are in fact dendritic cells. Again, I've found totally different reports on how IL-4 affects BMDC cultures, and even subsequent LPS stimulation, so I'm not even sure what I should be expecting.
Sorry for the long read, and thanks in advance,
No replies to this topic