Hi everyone,
I am doing osteoclast stimulation from RAW cell, passage 5. I seeded 2500, 5000 and 10000 cells in 96 well-plate with RANKL from R&D (20 and 40ng/mL), MCSF (25ng/mL) and change the medium every 3 days. The medium I used is alpha MEM with P/S and heat-inactivated FBS. But now is day 6, I could not find any giant cells in RANKL-treated group.
I would like to ask you what I should do or improve now? Or I just wait for a longer time.
Thank you in advance. I am looking forward to hearing from you.
Best regards,
Thuong
Hi Thuong,
There is no need to add m-CSF for differentiating RAW264.7 cells. RANKL alone is enough.
ATCC recommends high glucose DMEM for these cells. FBS is also very important factor for differentiating these cells.
I had previously answered this question. Check my recommendations here,
https://www.researchgate.net/post/Are_these_osteoclasts_Why_they_are_samller_and_fewer_than_literatures
Best,
Vishwa
Dear Vishwa Deepak,
Thank for your suggestion. I will try again with your recommendations.
Kind regards,
Thuong