Moghe:MDM: Difference between revisions
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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]] | ||
*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 | ||
* | *Add 35mL of cell suspension to BD T-175 tissue culture flasks | ||
*Let monocytes adhere | *Let monocytes adhere overnight in 37°C 5%CO<sub>2</sub> incubator | ||
*Wash cells | *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 | ||
*Replace media with M-CSF supplemented RPMI every 2-3 days | *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 96 well plates at 15,000 cell/well | |||
===Macrophage validation=== | ===Macrophage validation=== | ||
Revision as of 08:20, 15 May 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
- Replace media with M-CSF supplemented RPMI every 2-3 days
- Macrophages will be ready after 7 days of culture
- Wash, scrape and replate macrophages in 96 well plates at 15,000 cell/well
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)
