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 2.85*10⁶cell/mL in RPMI
• Add 35mL of cell suspension to BD T-175 tissue culture flasks
• Let monocytes adhere overnight in 37°C 5%CO₂ incubator
• Wash off non-adherent cells with PBS
• Add RPMI supplemented with 50ng/mL M-CSF and return to incubator
• Macrophages will be ready after 7 days of culture
• Wash, scrape and replate macrophages in well plates at 50,000 cell/cm²

Macrophage validation

• Cells should have high cytoplasm/nucleus size ratio
• Stain cells for several key markers of macrophages, immature monocytes and dendritic cells
  • Should express CD68, SRA1, CD36, EMR1, CD86, CD163, CD206 and CCR7 (macrophage markers)
  • Moderate CD14 expression (monocyte markers)
  • No CD1a and CD83 (dendritic cell markers)