I tried various protocols to differentiate RAW to clasts: I seeded 1,000 - 5,000 cells/well in a 96 well plate (the best results were with 1,000), used 150-200ng/ml mouse RANKL (didn't make a difference), DMEM High / MEMa (DMEM high was better).
The TRAP stain was positive and I was able to see the osteoclasts clearly as giant, multinucleated cells. However, I wasn't able to verify the differentiation. I tried RT-PCR for TRAP, CathepsinK, b3Integrin, MMP9 - didn't get any expression. I tried iNOS assay (Griess) and it was negative. I haven't tried quantitve TRAP Assay yet.
How can I be sure that my protocol is good and I get "real" osteoclasts?
Hello Tal,
I read it now. I imagine you have found a solution... I just want to tell you that with 30ng/ul RANKL is enough to differentiate osteoclasts from RAW. I work with a density of 25,000 cells/cm2. You dont need to spend so much. And after 6-7 days you have real osteoclasts that expressed TRAP, CTSK, MMP9, RANK...
Good luck!!!
Susana
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