Human osteoclast precursors (Lonza Walkersville, Inc.) were incubated at 37°C in 5% CO2 with CSF-1 (33 ng/mL) and RANKL (66 ng/mL) in the presence of either DMSO (0.2%) or PLX3397 (0.2% to maximum of 10 μM) for 6 days. The cultures were given fresh growth media and further incubated for 1 day. The cell supernatants were analyzed using the Acid Phosphatase Assay (Cayman Chemical) per manufacturer’s recommended procedure 35 (link). Absorbance was read at 405 nm on a Tecan Safire plate reader.
Human osteoclast precursors
Manufactured by Lonza
Human osteoclast precursors are primary cells derived from human peripheral blood mononuclear cells. They serve as a model system for the study of osteoclast formation and function.
Automatically generated - may contain errors
Lab products found in correlation
Safire, by Tecan (1 mentions)
Thp 1, by American Type Culture Collection (1 mentions)
M nfs 60, by American Type Culture Collection (1 mentions)
Tib 202, by American Type Culture Collection (1 mentions)
Mycoalert detection kit, by Lonza (1 mentions)
75 cm2 culture flask, by Corning (1 mentions)
M csf, by Merck Group (1 mentions)
Rankl, by Merck Group (1 mentions)
Accutase solution, by Merck Group (1 mentions)
3 protocols using human osteoclast precursors
1
Osteoclast Differentiation Assay
2
Culturing Human and Mouse Leukemia Cell Lines
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THP-1 (male human acute monocytic leukemia, #TIB-202; RRID:CVCL_0006) and M-NFS-60 (mouse myelogenous leukemia, #CRL-1838; RRID:CVCL_3543) were purchased from the ATCC. Cells were cultured in RPMI1640 media containing 10% FBS, 1% penicillin-streptomycin-L-glutamine, and 0.05 mmol/L 2-mercaptoethanol. M-NFS-60 cells were grown with 20 ng/mL mouse CSF1 (R&D Systems, #416-ML/CF). Cell lines were expanded upon receipt and frozen in aliquots at an early passage number. Cells were then passaged <6 months after resuscitation. The ATCC performs short tandem repeat (STR) analysis for characterization. Further STR characterization was not performed. Mycoplasma testing was performed monthly using the MycoAlert Detection Kit (Lonza, Inc, #LT07-318). Human osteoclast precursors were obtained from Lonza, Inc. (#2T-110).
3
Osteoblast-Osteoclast Co-Culture Protocol
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Commercially available human osteoblast cells (PromoCell) and human osteoclast precursors (Lonza) were cultured in their respective culture media (PromoCell and Lonza) at 37 °C with 5% CO2, and the media were replaced with fresh media every three days. The osteoblasts were passaged when they reached 70–80% confluence in a 75 cm2 culture flask (Corning, New York, NY, USA) using Accutase solution (Sigma-Aldrich, St. Louis, MO, USA). Osteoblasts from passages 4 to 7 were used in the experiments and seeded onto appropriate plates at a density of 2 × 104 cells/cm2. The osteoclast precursors were seeded onto appropriate plates for each experiment at a density of 4 × 104 cells/cm2, and human receptor activators of nuclear factor kappa-B ligand (RANKL; 40 ng/mL, Sigma-Aldrich) and human macrophage colony stimulating factor (M-CSF; 25 ng/mL, Sigma-Aldrich) were added to induce osteoclast differentiation.
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