Moghe:MDM: Difference between revisions
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*Wash off non-adherent cells with PBS | *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 | ||
*Macrophages will be ready after 7 days of culture | *Macrophages will be ready after 7 days of culture | ||
*Wash, scrape and replate macrophages in | *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 | ||
*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 | **Should express CD68, SRA1, CD36, EMR1, CD86, CD163, CD206 and CCR7 (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) | ||
Latest revision as of 09:04, 6 September 2012
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)
