Moghe:MDM
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(Difference between revisions)
| Line 21: | Line 21: | ||
===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 | + | *Stain cells for several key markers of macrophages, immature monocytes and dendritic cells |
| - | **Should have high CD68 | + | **Should have high CD68 and CD11b (macrophage markers) |
| + | **Moderate CD14 expression (monocyte markers) | ||
| + | **No CD1a and CD83 (dendritic cell markers) | ||
Revision as of 18:19, 17 October 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) or 24 (660μL) well flat bottom tissue culture plates
- Let monocytes adhere for 2-4 hours in 37°C 5%CO2 incubator
- Wash cells 3 times with PBS
- 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 and CD11b (macrophage markers)
- Moderate CD14 expression (monocyte markers)
- No CD1a and CD83 (dendritic cell markers)
