About
Primary monocyte differentiation to DCs with GM-CSF and IL-4
Notes
GM-CSF stock at 100ug/ml in PBS IL-4 stock at 100ug/ml in PBS
Method
Prepare differentiation media for target differentiation subset, in 50ml total volume RPMI 1640 + 1% GPS + 10% Hi FCS.
DCs add 12.5ul GM-CSF, 12.5ul IL-4
Transfer cells to 50ml tube and pellet by centrifugation 1200rpm 6 minutes
Resuspend cells in media for target subset at 1x106 cells/ml. e.g. To plate in 6 well plate, cells should be seeded at 3x106 cells/well in 2ml total volume.
Plate out cells or transfer to 25cm2 flask to culture. Incubate for 2 days.
Replace 2ml media by tilting plate carefully, and removing supernatant at edge. Note that cells will not have fully adhered at this timepoint. Incubate further 2 days.
Cells should have fully differentiated. Replace 2ml media again for experiment.