BM cells was harvested from B6, cultured in RPMI 1640 or DEME with 20ng/ml GM-CSF and 20ng/ml IL-4 for 6 days (seeding density was 1x10^6), 1μg/ml LPS was added on day 7, however, adherent cells were seem less than 5% along the way. Cells will be utilized for mix lymphocytes reaction experiments.
Hi Alexa:
It is well known that mature DCs have a different morphology than immature DCs (while iDCs are quite "round", mDCs are rather "spiculated"). However, in my experience this is not a characteristic you can explore in order to describe "DCs activation".
I would suggest you to analyse the expression of cell surface proteins (MHC-II, CD80/CD86, CD40, CD83, CCR7, etc), the production of cytokines (IL-12, IL-6, TNFa, among others), and/or the decrease in the phagocytic activity.
Additionally, while it is mandatory to use a combination of GM-CSF and IL-4 in order to produce "HUMAN" DCs, this is not an obligation in "MURINE" DC production (you can use GM-CSF alone).
Good luck!
Alexa:
For murine BM-DCs, the medium composition we use is RPMI-1640, FCS 10%, Pen/Strep (100 UI/ml and 100 ug/ml, respectively), Glutamine (2 mM), Hepes (10 mM), and 2-ME (50 uM). A key tip in DC culture is the FCS: low quality FCS will cause you troubles, so try to use the best one you can afford.
After activation, mDCs will attach to the plastic well; in order to detach these cells, try sterile cold PBS + 5 mM EDTA (5-10' incubation).
Good luck!
Hi Matías,
I got four more questions about BM-DCs:
1, Is it necessary to do RBC lysis right after BM harvest? If so, at what concentration?
2,Adherent cells are increasingly seen during the first 6 days(without LPS, GM-CSF alone), do they considered to be mature BM-DCs activated by repeatedly pipetting or other operation?
3,Will 1' incubation with trypsin do instead of EDTA?
4, I'm using radiated BM-DCs to do mix lymphocyte reaction experiment, but unfortunately stuck by the condition that massive apoptosis observed after radiation(25Gy), any suggestions?
Thanks!
Alexa:
I will try to answer your questions:
1. I perform RBC lysis employing the standard procedure described in "Current Protocols in Immunology".
2. The adherent cells that you see are probably from different populations than DCs. The original protocol of DC culturing included a step employing specific Abs and complement to lyse these populations, but I never did this. I just keep the "non-adherent or loosely adherent cells", that are up to 90% CD11c+.
3. I would not use trypsin, in order to avoid the enzymatic modification of cell surface proteins (that you may want to analyze by flow cytometry). Instead, I would just employ cold PBS + gentle pipetting to detach and recover mDCs after stimulation. Remember that if you use cold PBS + 5 mM EDTA + pipetting, you will get better results.
4. I tried twice to perform MLRs but had some troubles. However, you can try mitomycin C treatment as an alternative strategy to irradiation. Despite this, a colleague that performed several MLR assays told me that, if your CD11c % is higher than 90-95%, you may skip the irradiation step, since only the allogeneic lymphocytes in your mixture will be the ones that proliferate (the proliferation of the "contaminating" cells in your DCs will be undetectable).
I hope that these advices help you.
Good luck!
Hi Alexa:
In my experience, mDCs (after activation) get attached to the plastic plate, and you need PBS + 5 mM EDTA + pipetting in order to detach them; nevertheless, some cells always remain attached even after this treatment.
You have to think about your experiments in order to decide what to do next:
a. if your aim is to measure the expression of some cell surface markers by flow cytometry, then you might increase a little bit the pipetting (since you are going to stain and fix these cells almost immediatly, this stronger conditions won't affect too much your activation status);
b. however, if your aim is to use these activated DCs in MLRs or to inject them into recipient mice, then I would prefer to loose some cells that remain attached, so I won't activate them by unspecific conditions like pipetting.
I hope this helps. Good luck!
