Moghe:MDM: Difference between revisions
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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 | *Plate in 96 (100μL) well flat bottom tissue culture plates | ||
*Let monocytes adhere for 2 | *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 | **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 14:38, 4 November 2010
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)
