I can not see any osteoclasts in my culture till day 7 and I am using RANKL from abcam (ab129136).
https://www.researchgate.net/post/My_raw2647_cells_are_not_differentiating_into_osteoclasts_using_abcams_RANKL-ab129136_Has_anyone_used_this_or_has_any_recommendations_for_diff
Anuruddhika Udayangani Rathnayake Thank you very much. I already tried this before but didn't work.
Please check your passage number (low passage number is recommended) and seeding density. Use phenol red free media supplemented with charcoal stripped serum. You need to give us the details of RANKL conc, passage number, osteoclast TRAP stained images etc. for efficient troubleshooting. Please go to the following link and check if you are facing the same issue. All the Best!
https://www.researchgate.net/post/How_to_find_and_quantify_osteoclast_differentiated_from_RAW_2647
Dear Shubhangi Gavali,
I have tested 3 different concentrations of RANKL concentrations (50, 75 and 100 ng/ml). I maintained the passage number below 10 and used 10000 cells /ml. Since I didn't observed any nuclear fusion or typical giant cells I didn't stained them.
Neelaka Molagoda, after ruling out RANKL conc and passage number of cells, I think you should try new aliquot of RANKL (or new vial altogether). There could be some issue related to its stability in current aliquots. Also, try troubleshooting on a newly revived batch of low passage cells.
One tip: Never discard OC culture (even the 'failed' one) without staining. At Least fix and stain the cells with hematoxylin and store the plate for reference. This will tremendously help you when you are comparing various protocols (for cell number, morphology, etc) applied for optimization.
Hope this helps. All the best!
Shubhangi Gavali Thank you very your tips and suggestions. I'll try to optimize my protocol along with your ideas too.
Cell density and passage numbers affect the differentiation. I have been using 25000-30000 cells per well in 24 wells plate with RANKL at 20ng/ml final concentration with 1ml media. Change media alternate day and you can see large osteoclast after 72hrs.
For 6 wells plate 400000 cells per well, 2ml media. For 6 wells plate format, I often get osteoclast after 72hrs and sometime I need to keep for 120hrs.
Good luck.
Anuj Anuj Thank you very much. I gave up the experiment but I will try one more time in your way.
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