Moghe:MDM

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)

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)