I'm following the protocol (https://www.frontiersin.org/articles/10.3389/fphar.2018.00071/full):
Cell differentiation was induced via a 6-h exposure to 185 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma–Aldrich) in DMSO. Cells were then polarized toward M1 or M2 phenotype by incubation for 48 more hours with INF-γ (20 ng/ml, Immunotools) and LPS (100 ng/ml, Sigma–Aldrich) or with IL-4 (20 ng/ml, Immunotools) and IL-13 (20 ng/ml, Immunotools), respectively (Tjiu et al., 2009; Maeß et al., 2014), still in the presence of PMA. Cells used for the resting condition were kept in the presence of PMA for 48 more hours in normal growth medium. CM were then prepared by keeping polarized as well as resting cells in serum-free RPMI without stimuli and no PMA for 72 more hours.
Basically, what I'm seeing are wells with M1 macrophages that dwindle in number over the 72 hours post-exposure to INF-γ and LPS. I'm not sure if this matters, but their distribution is typically only at the periphery of the well by the end of that time (images attached are of this area). Also, for some reason, their association (phagocytosis) with beads is lower than both M0 and M2. What could I be doing wrong?
Troubleshooting steps:
-Order fresh reagents & prepare new aliquots
-Tried another vendor
Dear Cody,
I observed the same issue with primary human monocyte-derived macrophages. For my primary cells, the cause of death was a prolong exposure to the polarizing cytokines namely LPS and IFNy (M2 polarization with IL-4 survive better). Hence, we moved away from exposing macrophages to the polarizing agents for more than 48h. The polarization M1 markers that I use to identify M1 macrophages remain highly expressed for several days without LPS/IFNg stimuli but the expression levels decreased over time. I perform my assay 7 days after polarization and I can still distinguish M1 from M2 macrophages, but M1 macrophages on day 1 without IFNg/LPS have higher expression of polarization markers compared to M1 macrophages on day 7. Survival signal M-CSF remains in the culture - without it, macrophages are dying.
Good luck with your experiments!
A comment on the phagocytosis: I have the same observation that M1 are phagocytosing less than M0 and M2 (phagocytosis of apoptotic cells)! I´m still puzzled since most literature doesn´t align with it. Let me know if you managed to answer this question.
Best,
Bianca
Dear Cody,
Try by reducing the LPS concentration and exposure time. Typically 24 to 48 hour is enough. High concentration of LPS or longer exposure time may result in cell death.