I've come across two ways to generate M2b macrophages from primary human monocytes.
Iqbal and Kumar, J Clin Cell Immunol 2015, 6:6 https://goo.gl/UQbq5X
M2b: 1ug/ml LPS + 10ng/ml IL-1beta https://goo.gl/B7vue7
and
Clin Immunol. 2014 May-Jun;152(1-2):10-9
M2b: 10 ng/ml LPS and 33 μl/1 ml medium immune complexes ( 1 μl 1.32 mg/ml BSA and 50 μl rabbit polyclonal anti-BSA were mixed and incubated for 1 h at 37 °C)
LPS is a constant for both methods. It comes down to IL-1b vs immune complex. Is there a consensus to which one is preferred?
Very simply, just prime them with LPS and IFN-gamma for one day and then put them with tumor lysate or IL-13 and u can also add chitosan particle or TLR4 ligand also and check the expression of YM-1 and FIZZ1 along with arginase-1
I don't like LPS because the phenotype of the resultant macrophages is that of a tolerized macrophage instead of a functional M2 type. I use IL4 (40ng/ml) + IL10 (50ng/ml) for 3 days followed by reestimulation with half of that amount for the next 4 days. At the end of the 7-day culture, you'll have completely differentiated macrophages with a high percentage of M2 (never 100%). human macrophages do not express the same makers than murine macrophages, so be careful with your selection of markers.
Best wishes.
Hi Paul,
M1-macrophages can be generated by addition of IFN-γ (50 ng/ml) and LPS (10 ng/ml). For M2 phenotype, M2a-activation can be achieved by 20 ng/ml IL-4. Supplementation with immune complexes and IL-1β or LPS will elicit M2b-activation, whilst IL-10, TGFβ or glucocorticoids lead to M2c-activation of M2-macrophages.
Culturing primary human monocytes with GM-CSF+IL-4/IL-13 will result in DC while M-CSF+IL4 will result in M2. Culturing the cells with M-CSF for 3 days and then with fresh M-CSF and IL-4 for an other 3 days will result in M2.
M2-macrophages are characterized as CD68+/CD163+ while M1 being CD68+/CD80+
BW
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