Hello!
I have work with osteoclasts obtained from RAW.
Now i would like to replicate my experiments in osteoclasts from PBMCs. I isolate monocytes and plated with a density of 250,000 cells/cm2. I treated with 30 ng/ml of hM-CSF and h-RANKL. But after 2 weeks i have not osteoclasts yet. It is normal?
Thank you very much in advance
Susana
Dear Susana Jurado
I briefly wanted to reach out for your inquiry. Please check if you have used filtrated or charcoal stripped FBS? The differences are quite remarkable as per Figure 1 of the attached publication.
Article An Optimized Method to Generate Human Active Osteoclasts Fro...
You may also want to titrate down your cell density to improve OC formation. Please check the paper for details as it is quite exhaustive in checking different parameters concerning the optimization for OC derived from PBMCs.
I hope that helps.
All the best & good luck experimenting,
Michael
Dear Michael Förster,
Thank you so much! I will read!
All the best
Susana
Dear @Susan, one of my friend differentiate monocytes in to osteoclasts. On 21 days of culture with periodically replenishment of factors, osteoclasts are seen.
I guess you should increase time and see.
All the best.
Thank you very much Jitendra Kumar Shandilya ! In the bibliography i have read in some articles that osteoclasts are seen after 7 days and in other that they need more than 21 days to differentiate.
Best regards!!
Susana