Moghe:MDM

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*[[Moghe:PBMC |Isolate mononuclear cells from buffy coats]]
*[[Moghe:PBMC |Isolate mononuclear cells from buffy coats]]
*Dilute cells to 2*10<sup>6</sup>cell/mL in RPMI
*Dilute cells to 2*10<sup>6</sup>cell/mL in RPMI
-
*Plate in 96 (100μL) or 24 (660μL) well flat bottom tissue culture plates
+
*Plate in 96 (100μL) well flat bottom tissue culture plates
-
*Let monocytes adhere for 2-4 hours in 37°C 5%CO<sub>2</sub> incubator
+
*Let monocytes adhere for 2 hours in 37°C 5%CO<sub>2</sub> incubator
-
*Wash cells 3 times with PBS
+
*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
*Add RPMI supplemented with 50ng/mL M-CSF and return to incubator
*Replace media with M-CSF supplemented RPMI every 2-3 days
*Replace media with M-CSF supplemented RPMI every 2-3 days
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*Cells should have high cytoplasm/nucleus size ratio
*Cells should have high cytoplasm/nucleus size ratio
*Stain cells for several key markers of macrophages, immature monocytes and dendritic cells
*Stain cells for several key markers of macrophages, immature monocytes and dendritic cells
-
**Should have high CD68 and CD11b (macrophage markers)
+
**Should have high CD68, SRA1, CD36 (macrophage markers)
**Moderate CD14 expression (monocyte markers)
**Moderate CD14 expression (monocyte markers)
**No CD1a and CD83 (dendritic cell markers)
**No CD1a and CD83 (dendritic cell markers)

Revision as of 16:38, 4 November 2010

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*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)
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