Hi, I am trying to set a co-culture system of T cells from immunenized mice and mcsf treated mouse bone marrow. However, it is not successful to induce osteoclasts. I read that we can fix stimulated lymphocytes by formalin before seeding osteoclast precursors onto. I am not sure it will work with fresh sorted T cells. In this case, we just remove medium and add formalin? I think it is impossible because T cell is not adherent for replacing medium by formalin.
Everyone has idea about it?
Thank you so much
Hi Nam,
For Osteoclast culture you have use MCSF and RANKL.First culture osteoclast from Bone marrow allow them from single nucleated state to multi-nucleated state.after 60-70 percent confluence.then you can add you T cells. for another 24 or 48 hrs as co-culture.
We used to incubate bone marrow just with RANKL on a treated plate. After two days or so only the adherent osteoclasts survived, and the rest just died and can be washed away easily.
And if you want T-cells to adhere to the plate for short co-incubation, centrifuging the plate at 400g for 4 minutes, should suffice.