Moghe:MDM

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Current revision (12:04, 6 September 2012) (view source)
 
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====All work should be done inside a cell culture hood====
====All work should be done inside a cell culture hood====
*[[Moghe:PBMC |Isolate mononuclear cells from buffy coats]]
*[[Moghe:PBMC |Isolate mononuclear cells from buffy coats]]
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*Dilute cells to 2*10<sup>6</sup>cell/mL in RPMI
+
*Dilute cells to 2.85*10<sup>6</sup>cell/mL in RPMI
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*Plate in 96 (100μL) or 24 (660μL) well flat bottom tissue culture plates
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*Add 35mL of cell suspension to BD T-175 tissue culture flasks
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*Let monocytes adhere for 2-4 hours in 37°C 5%CO<sub>2</sub> incubator
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*Let monocytes adhere overnight in 37°C 5%CO<sub>2</sub> incubator
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*Wash cells 3 times with PBS
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*Wash off non-adherent cells with PBS  
*Add RPMI supplemented with 50ng/mL M-CSF and return to incubator
*Add RPMI supplemented with 50ng/mL M-CSF and return to incubator
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*Replace media with M-CSF supplemented RPMI every 2-3 days
 
*Macrophages will be ready after 7 days of culture
*Macrophages will be ready after 7 days of culture
 +
*Wash, scrape and replate macrophages in well plates at 50,000 cell/cm<sup>2</sup>
===Macrophage validation===
===Macrophage validation===
*Cells should have high cytoplasm/nucleus size ratio
*Cells should have high cytoplasm/nucleus size ratio
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*Stain cells for several key markers of macrophages and immature monocytes
+
*Stain cells for several key markers of macrophages, immature monocytes and dendritic cells
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**Should have high CD68, some CD14, no CD1a
+
**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)

Current revision

Contents

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)
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