Hello,
I have a question regarding the differentiation of primary monocytes.
I have isloated monocytes from PBMCs and would like to differentiate them into M1 macrophages. Does anyone have a protocol on how much GM-CSF is needed, how long the differentiation takes and how often you have to change the medium with GM-CSF?
I would be grateful for any suggestions!
Hello Kiku Takeuchi
Please refer to the attached article in the Protocol section under
Differentiation and polarization of monocyte-derived cells.
https://www.jove.com/pdf/61807/jove-protocol-61807-polarization-m1-m2-human-monocyte-derived-cells-analysis-with-flow
For macrophage differentiation, prepare a working solution containing 50 ng/mL GM-CSF or M-CSF for M1 and M2 macrophage polarization respectively, added in 2 mL of RPMI complete medium per well. Culture the cells in a 5% CO2 incubator in 37 °C for 3 days……….
Best.
Isolated monocytes were seeded at a density of 105 cells in 12 well plates, where Poly-D-lysine (PDL)coated 18 mm round coverslips were already placed. For differentiation into macrophages of M2 (anti-inflammatory) phenotype, 500 µl of complete RPMI 1640 was supplemented with 100 ng/ml of M-CSF (Macrophage- colony-stimulating factor). Cells were incubated in M-CSF for 24 hrs at 37ºC in 5% CO2, to let adhere monocytes on the PDL-coated coverslips in 12 well culture plates. After 24 hrs of incubation non-adhered cells were removed by removing the media from wells and adhered cells were washed twice with 1X PBS to remove any remaining non-adherent cells. Further, cells were incubated for 7 days at 37ºC in 5% CO2 for complete maturation of macrophages. The medium was replaced after every 48 hrs of incubation with the washing of cells twice in 1X PBS. Macrophages were assessed for the development of characteristic structure on 1, 3, 5 and 7 days via fluorescence staining of CD163.
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