I can not see any osteoclasts in my culture till now. I have done a TRAP assay twice, but there is no sign of any osteoclast after 7 days. The media was changed every three days. Please help me if anyone knows how to troubleshoot it. It's well within 20 passages.
http://www.ncbi.nlm.nih.gov/pubmed/22130930
http://www.sciencedirect.com/science/article/pii/S1534580702003696
Maybe these can help...
But I wonder why u r not isolating directly from the bones (I dont know if that is possible!!!)?
We need huge number of osteoclasts for some biochemical work. We are doing primary osteoclast isolation and differentiating from bonemarrow also, but somehow we are not yet able to get a large number of osteoclasts. Generally from the literature and from other threads I learned that RAW cells generally gives a large number of osteoclasts in about 5-6 days when cultured with RANKL. Thank you for your input. I have already looked at Osdoby's paper, but it is to some extent confusing if you're a newcomer, though I have set up the basic culture protocol from them only.
I would check for the inhibitors that are reported and see whether my culture conditions contain them, by chance or mistake. In cell culture, with my limited knowledge, i know there are many times where 1/2 inhibitors might play spoilsport. So see if u can get them... I hope some expert in the field might answer soon... :)
Once you have prepared your RANKL solution, should be used within 4–8 weeks, even if it has been aliquoted and stored at -20.
The key is often the correct density. RAW cells don't form osteoclasts if they are seeded to dense. The optimum I obtained for this cell line in our lab is 12500cells/cm². Stimulated with 10-20ng/ml RANKL (directly with seeding) they form osteoclasts within 72-96h.
Thank to both of you Juliane and Xiong, I will do exactly like you said and let you know what happens.
Do you use mouse or human RANKL from R&D? For the human one it is recommended the use of a cross linking antibody.
Hi Arijit, not sure if you were able to troubleshoot yet.
For RAW264.7 macrophage to osteoclast differentiation 4 things are critical.
1) seeding density 2) passage number 3) good quality RANKL 4) RANKL concentration.
1) If you seed in a 96 well plate (saves RANKL) seed 5000 cells/well.
2) In our experience RAWs with passage number higher than 10-12 start to loose differentiation potential. Additionally never grow them to over-confluency. They won't differentiate even at low passage.
3) R&D RANKL is good. But better buy with a carrier such as BSA.
4) I have tried R&D RANKL as low as 10 ng/ml with excellent results. Ideally we use 15ng/ml RANKL for routine work.
If you follow all the aforementioned you will see differentiation from 3rd day and giant osteoclasts by 5th day.
PS: Closely record the morphology of RAWs as shown on ATCC website. In case you notice a change in morphology, discard the cells in culture and grow new stock.
Good luck
Hi Viswa,
Thanks for helping. My problem is almost solved, meaning, I get osteoclasts, but not "Giant" ones, I usually seed 25000 RAW cells in 24 well plate. Is that a problem?
Regards,
Arijit
We seed 15 000 / 20 000 cells in 24 well plate for OSC differentiation. See which one works for you. Higher seeding density may lead to contact inhibition.
Good luck
Dear Arijith
Osteoclastogensis mainly depend on the
number of cells seeded in the plate : 1.5 x 105/cm2
passage number of raw cells : dont use cells with more than 18-20 passage number
Concentration and source of RANKL: human or mouse RANKL at 50-100ng/ml
enzo life/ invitrogen/promokine is gud
Seeding density is very important. I am getting osteoclasts by seeding 10K Raw cells in 24 well setup with RANKL (30ng/ml)
I also confirm using 1.5 X10^4 cell/ml for 24 well plates and 15 ng/ml of RANKL.
RAW 264.7 TIB-71 (ATCC) is this the catalog number for cell lines that can differentiate osteoclast from RAW cells?
Yes. That's the correct cell line. Be aware that it is S2 biosafety level and as you read here there are a lot of things that need to be optimized so that it really works. Good luck!
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