Dear all,
We have some problems with generating osteoclasts out of murine bone marrow cells. So I would like to ask in general:
- Do you pretreat the cells before you culture them?
- Which medium do you use (company, + 10 % FCS? + 1 % Pen/Strep?)
- Which cytokines etc. (M-CSF, RANKL, Vit D, ascorbic acid...) do you supplement and which concentration do you use?
- From which company do you purchase the medium/ cytokines etc.?
- How long do you culture the cells?
I would be glad if somebody could tell me how you manage to generate your osteoclasts.
Best regards.
Hi Anja,
A colleague of mine had some considerable success with isolation and culture of osteoclasts, Unfortunately I cant remember all of the media details but I understand he used M-CSF, RANKL and I think (but not 100%) the base medium was RPMI . If you private message me I will pass his details to you so that you can ask him for help if you like?
Best wishes,
Joe
Hi Anja,
in our lab we are used to culture Osteoclast from murine bone marrow. After flushing whole bone marrow, just plate it at 4*10 5 cells/cm2 in alpha MEM (gibco or Sigma are fine) + 10%FCS (gibco or Sigma are fine) + 1 % Pen/Strep + 10 ng/ml MCSF (R&D system) + 10 ng/ml RANK-L (R&D system). If you are culturing OCs from "supposed" osteoporotic mice (aged, ko, and so on), you will not have to change the madium, because you might find multinucleated cells already after 3 days. If you are treating bone marrow from normal mice (young, wt, ...), you will have to change the medium after 3 days. I suggest to change the medium early in the morning, because after the first change of medium you will see the first multinucleated cells already after 3-4 hours and, they will grow so fast that you could lost them in 12-24. So stop or fix them by 12 hours from the medium change.
My bests,
graziana
Dear Graziana,
Thanks a lot for your advice. However, I still have a few questions. Does it matter with batch of FCS you use? Since we had to change the FCS we didn’t manage to generate OCs again. And I read in some other fora that it makes a huge difference which batch you use. Did you have similar problems? Since there are a lot of different FCSs and a-MEMs (with or without nucleosides, with L-Glutamine or GlutaMAX etc.) could you possibly tell me the order no.? And in case of the RANKL (by R&D systems), do you use the one expressed by E. coli or the one derived from a mouse cell line? Again, thank you very, very much for your help!! Anja
Dear Anja, yes you are right! Sometime the different batch of FCS can influence the OCs formation. We use a-MEM with nucleosides and L-Glutamine (order no. M 0644). RANKL is that one expressed by E. coli.
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