PBMCs isolated by density gradient centrifugation were plated in 96-well culture plates as described before.19 (link) PBMCs were left overnight for osteoclast precursors (OCPs) to adhere on bone slices (Immunodiagnostic Systems, Boldon, UK).20 (link) On the following day (day 1 of culture), medium was changed to Dulbecco's modified Eagle's Medium (DMEM) supplemented with macrophage colony stimulating factors (M-CSF) 25 ng/mL (Peprotech, London, UK). Three days later, medium was again changed to DMEM with M-CSF (25 ng/mL), soluble RANKL (sRANKL) (50 ng/mL; Peprotech), dexamethasone (10 nM; Sigma-Aldrich) and transforming growth factor β (2.5 ng/mL; R&D Systems) in order to differentiate the OCPs into mature OC.21 (link) The culture medium was then changed twice a week. Cells cultured on bone slices for 7, 14 and 21 days20 22 (link) were used for functional assays and gene expression.
Protocol detail
Osteoclast Differentiation from PBMCs
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Assays
Bone
Cells
Culture medium
Density gradient centrifugation
Dexamethasone
Gene expression
Immunodiagnostic
Macrophage colony stimulating factors
Osteoclast
Rankl
Transforming growth factor β 2
Corresponding Organization : University of Lisbon
Other organizations : Centro Hospitalar Lisboa Norte, Hospital de Santa Maria, Universidade Nova de Lisboa, University of Helsinki
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Variable analysis
independent variables
- Duration of cell culture (7, 14, and 21 days)
- Functional assays
- Gene expression
- PBMCs isolated by density gradient centrifugation
- PBMCs plated in 96-well culture plates
- PBMCs left overnight for osteoclast precursors (OCPs) to adhere on bone slices
- Medium changed to DMEM supplemented with M-CSF (25 ng/mL) on day 1 of culture
- Medium changed to DMEM with M-CSF (25 ng/mL), sRANKL (50 ng/mL), dexamethasone (10 nM), and transforming growth factor β (2.5 ng/mL) to differentiate OCPs into mature osteoclasts
- Culture medium changed twice a week
- Positive control: Not specified
- Negative control: Not specified
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