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Materials

  • PBMC
  • α-MEM culture medium containing 10% FBS
  • M-CSF
  • TGFβ
  • RANK-L

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.

Procedure

  1. Thaw PBMC and suspend in α-MEM containing 10% FBS
  2. Count cells and adjust concentration to 3 x 105 per mL in α-MEM, 10% FBS
  3. Add M-CSF to a final concentration of 25 ng/mL and TGFβ to a final concentration of 5 ng/nL
  4. Add RANK-L to a final concentration of 50 ng/mL
  5. Add cells to a 96-well, flat-bottom plate at 200 uL of cells per well (60,000 PBMC per well)
  6. Feed cells every 3–4 days by removing 100 uL of medium and replacing with 100 uL of fresh medium containing growth factors