Moghe:MDM

Differentiation of Macrophages from PBMC monocytes

Materials

• Sterile 50mL conical tubes
• 24 or 96 well flat bottom tissue culture plates
• Sterile PBS
• RPMI 1640 (10% FBS, 1%pen/strep)
• Human M-CSF (Peprotech)

Procedure

All work should be done inside a cell culture hood

• Isolate mononuclear cells from buffy coats
• Dilute cells to 2*10⁶cell/mL in RPMI
• Plate in 96 (100μL) well flat bottom tissue culture plates
• Let monocytes adhere for 2 hours in 37°C 5%CO₂ incubator
• Wash cells 3 times with PBS (use multichannel pipette to gently remove and replace)
• Add RPMI supplemented with 50ng/mL M-CSF and return to incubator
• Replace media with M-CSF supplemented RPMI every 2-3 days
• Macrophages will be ready after 7 days of culture

Macrophage validation

• Cells should have high cytoplasm/nucleus size ratio
• Stain cells for several key markers of macrophages, immature monocytes and dendritic cells
  • Should have high CD68, SRA1, CD36 (macrophage markers)
  • Moderate CD14 expression (monocyte markers)
  • No CD1a and CD83 (dendritic cell markers)