Profiling M1 and M2 Macrophage Phenotypes

RNA was isolated from DAPI¯/ CD11b⁺/ F4/80⁺ phenotype cells sorted using FACS AriaIII apparatus (BD). Total RNA samples were prepared using the Total RNA Mini Plus kit (A&A Biotechnology, Gdynia, Poland), according to manufacturer’s procedure. cDNA was synthesized with M-MuLV reverse transcriptase and random hexamers (EURx, Gdańsk, Poland). Starters characteristic for M1 and M2-specific cytokines and chemokines genes were employed (mArg-1, mCCL-22, mMRC-1, mCCL-17, mCSF-1, mMMP-9, mIL-10, mCXCL-11, miNOS, mIL-1b, mCXCL-9, mIL-12a, acc. to Huang et al. [36 (link)]; mVEGFa, mPlGF [57 (link)]; mVEGFc and mNRP-1 [58 (link)]). mRNA levels’ measurements were performed by SG qPCR Master Mix (2x) (EURx, Gdańsk, Poland). The amplification was performed using a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). Relative quantitation of mRNA was performed using ΔΔC_T method with β-actin as a reference gene. Means were calculated from three independent repeats.