First experiment: raw264.7 cells induced osteoclast differentiation, M-CSF (30 ng/ml) + RANKL (100 ng/ml), changed the medium every two days, no osteoclasts appeared on the 7th day of induction.
Second experiment: After taking out at -20℃, mouse primary macrophages (bone marrow source) were induced with M-CSF (30 ng/ml), and different concentrations of RANKL (50~100 ng/ml) were added on the third day. After adding RANKL, the culture was continued for 7 days, and the macrophages were polarized, but no osteoclasts were seen.
Any suggestion would be greatly appreciated!
Marine Ma ,
1. It is recommended to increase the pretreatment of Raw264.7 cells. Primary macrophages derived from bone marrow need to be pretreated with M-CSF for 5-7 days (not 3 days) to fully differentiate into precursor cells with osteoclast differentiation potential (CD11b⁺F4/80⁺). If the pretreatment time is too short, the cells may still be in an immature state and unable to respond to RANKL signals.
Recommendation: After primary macrophages are isolated, they are first cultured with M-CSF (30-50ng/ml) for 5 days, and the cell morphology (round or irregular shape, adherent growth) is observed every day. RANKL is added when the cell density reaches 70%-80%; the expression of CD11b and F4/80 can be detected by flow cytometry to confirm the purity of macrophages (should be > 80%).
2. Appropriately increase the RANKL concentration to 150ng/ml; if there is still no improvement, further detection of cell surface RANK receptor expression (flow cytometry detection of RANK) is required to rule out receptor deficiency.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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