Human monocytes can be cultured to create many other cell types. This protocol was designed to fine-tune previously published methods for differentiating human monocytes into osteoclasts — macrophages found in bone tissue.
- Thaw PBMC and suspend in α-MEM containing 10% FBS
- Count cells and adjust concentration to 3 x 105 per mL in α-MEM, 10% FBS
- Add M-CSF to a final concentration of 25 ng/mL and TGFβ to a final concentration of 5 ng/nL
- Add RANK-L to a final concentration of 50 ng/mL
- Add cells to a 96-well, flat-bottom plate at 200 uL of cells per well (60,000 PBMC per well)
- Feed cells every 3–4 days by removing 100 uL of medium and replacing with 100 uL of fresh medium containing growth factors