Hi! I am trying to polarize RAW 264.7 macrophages on the 7th pass (ATCC) with 100 ng/mL LPS (Sigma) for M1 and 40 ng/mL IL4 (Sigma) for M2. They were plated at a density of 20,000 cells per well, allowed to stabilize for 24h and exposed to LPS and IL4 for 1h.
When analyzing by qPCR, I found no difference in gene expression of any of the following genes: TNF A; ARG1; IL1B; iNOS; HO1; NRF2 and GMCSF.
These are protocols established in the literature and were followed to the letter, but I could not prove the polarization process.
Should I change markers? Was the exposure time to LPS and IL4 short? Concentration should be higher?
Thanks!
Regards,
Antonio.
Hi Antonio,
It's been a while since I've worked with RAW264.7 (6 years ago), but at that time I used to use LPS 10ng/mL + IFNγ 10ng/mL and evaluated the supernatant after 24h to see M1-related cytokine production.
In your case, your protocol seems okay, but maybe if you treat cells longer you may start to see differences. You can try a time-point kinetic (4h, 8h, and overnight), perhaps you find an optimal time-point for your system.
Best,
Rhaissa
Hi Antônio Secco Martorano , If you thinking about the makers of M1 and M2 polarization, This paper will give you a clear idea in choosing markers (Novel Markers to Delineate Murine M1 and M2 Macrophages; DOI: 10.1371/journal.pone.0145342). According to them, TNF a, Arg1, and IL-1B are not distinctive markers (Fig2). You can try some other distinctive markers such as Fpr2, Gpr18, CD38 for M1 and c-Myc, Egr2 for M1. In one of my publications, I have used them and I used M1/M2 gene expression ratios to delineate the activation state (Cytosolic β-catenin is involved in macrophage M2 activation and antiviral defense in teleosts: Delineation through molecular characterization of β-catenin homolog from redlip mullet (Planiliza haematocheila).
Hi Rhaissa Vieira , i will try to increase the exposure periods. I also performed an assay with 4h exposure (200 ng/mL for LPS and 40 ng/mL for M1 and M2 polarization) and the iNOS and Arg1 genes did not show differences in qPCR.
Thanks a lot for the help!
Hi Thilina Kasthuriarachchi , i looked at the paper you recommended, but I'm not sure if these markers are expressed by RAW 264.7, because in the study they used bone marrow derived macrophages. I'll see if I can find anything related. Thanks a lot for the help!