Hi,

I tried osteoclast differentiation with mouse bone marrow cells a few times, and while they are positive for TRAP staining, I do not see any mature osteoclasts with 3 or more nuclei.

I have tried plating total bone marrow, PBMC after density gradient centrifugation, and CD11b+ cells from PBMC. I cultured them in 10% FBS DMEM with M-CSF (30ng/mL) for 5 days and then add RANKL (50ng/mL) for 10 - 14 days, media is changed every 2-3 days.

TRAP is completely negative in M-CSF only condition, and qPCR shows several hundred-fold increases of Ctsk and Acp5 so I think the cells are differentiated. But I don't ever see multi-nucleated huge cells that are shown in many papers. I only see small cells with one or occasionally two nuclei that are TRAP positive. There are a few bigger cells with very strong TRAP staining but I can't see the nuclei due to staining, and they are rare to find. For TRAP staining, I used Sigma TRAP kit (Cells are fixed in 4% PFA for 10 min, permeabilized in 0.1% triton x-100 for 1 min before the staining)

I am now trying RANKL (100ng/mL) but I don't see much morphological difference after 5 days.

Also, should I NOT permeabilize the cells before TRAP staining?

Any suggestion would be appreciated! Thank you for reading.