Does anyone culture pDCs from murine bone marrow cells? I am getting very low yield of pDCs from BM cells.
From the hind limbs I get an average of 50 million cells/mouse to start with. I do a 8 day culture in ACCM with Flt3, replenishing the media on day 4. On day 8 I harvest the cells and get around 8 million cells per mice, after MACS sorting the cells with B220 beads I get only an average of 0.4 million B220+ cells per mouse.
Is there anyway to increase the yield?
Use IMDM or RPMI, add beta mercaptoethanol if you haven't already (regular concentration for culture), and batch test several FCS. If that doesn't work let me know. My protocol is attached but misses the crucial instruction for beta mercapto
Hi,
you need to culture BM cells at the concentration of 1.5 million cells per mL in RPMI 10%FBS, 1%P/S, glutamine, beta mercaptoethanol and FLT3L. You can culture them in 24well plate changing half of the medium at day 4. At day 8 you will get 70% of pDCs (siglecH+ CD11c+). The B220 marker is not the best choice for MACS sorting.
Julien
Also, you can try adding some M-CSF to the FL cultures. See this paper
http://www.ncbi.nlm.nih.gov/pubmed/19941113
Julien's advice for culture density is correct, although you can go as high as 3 mill/mL, and in larger vessels, depending on your purpose.
Thanks Shalin and Julien for the advice. What is the concentration of 2ME that you usually add in the culture media? I use less than 10 micromolars. Also other than using B220+ MACS seperation which is the best way to sort the pDCs from the culture media on day 8, since the aim of the experiment is to stimulate the pDCs with various ligands. Have anyone done any stimulation experiments before with pDCs? If so, what is the minimum concentration of the cells used per well to get detectable levels of cytokines? I have done several batches of cultures but since I have low numbers of pDCs from each mice, not sufficient to do multiple stimulants.
CD45RA is a great marker. Also mPDCA-1 from miltenyi. I would culture 1x10e5 per well for stimulation. Then you only need a fraction of that supernatant for ELISAs etc (see my Naik, Proietto JI paper for conditions, or anything by Meredith O'Keeffe). I see that I add 28.4 µM 2Me (I don't know why it's such a weird concentration, but it's not far off your 10 µM so your conditions should work). Are you maximising harvest from bones? Progenitors are mostly in the epiphysis so you need to make sure you don't cut them off in your preparation. good luck.
- Badges
- Science topic
- Similar topics
- Suckling Animals
- Eutheria
- Rodentia
- Muridae