Before purchasing abcam's RANKL (ab129136), I contacted the company as to whether a crosslinking antibody was necessary because I didn't want to use one. They assured me that I did not need one. But despite using the protein at up to 10x's the recommended dosage, I still cannot get my Raw264.7 cells to differentiate into osteoclasts.
My cells grow VERY fast. Despite splitting them at the highest recommended ratio (1:6), I still need to split them every 2 to 3 days to prevent them from growing beyond 60-70% confluence. I am trying to use them at as low a passage number as possible since they have a strong tendency for drifting. Visually, they seem to look good (ie: similar to images I've seen ECACC's website).
I am new to this field and have never worked with Raw cells or RANKL before and am not sure whether my problem is with the cells or the RANKL protein.
Any help would be appreciated.
I also use soluble RANKL from Peprotech and I have found that 50ng/ml is a good concentration when growing 2x 105 cells per well in 48 well plate. When I was looking for literature on this, I could see that most of the people use RANKL either from Peprotech or R&D and I chose the first option due to difference in price but both should work equally good.
What is the passage number of your RAW 264.7 cells? Too high a passage number will affect differentiation to osteoclasts. For us, after 15-20 passages the cells become irregular and differentiation to osteoclasts is affected.
Dear Tia were u able to get the mature OC? I am still struggling with this system. Can any one help?
Regards
Many factors affect the differentiation potential of RAW264.7 into osteoclasts for instance, the passage number, ligand dose, duration of treatment and type of cell culture media. Use cells upto 15 passage, try to keep the 50ng/ml to 100ng/ml dose of ligand, grow cells for atleast 7 days. If still it is not working then there is a certain problem with the RANK ligand that you are using
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