2.10. qPCR Analysis of M1 and M2 Macrophage Markers

Total RNA from BMDMs was extracted using the TRIzol™ Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA was synthesized using 1 µg of RNA with the SuperScript III Reverse Transcriptase (Invitrogen), according to the manufacturer’s instructions. Real-time PCR was performed in duplicate, with obtained cDNA, specific primers and Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), using the StepOne™ System (Applied Biosystems). The data were analyzed using StepOne™ System software with a cycle threshold (Ct) in the linear range of amplification and then processed by the 2^–ΔΔCt method. The dissociation step was always included to confirm the absence of unspecific products. GAPDH was used as an endogenous control to normalize the variability in expression levels and results were expressed as fold increase. Expression levels for Arginase-1, mannose-receptor (CD206) and iNOS were calculated. Primers (IDT) used were as follows: iNOS (5′-AGCACTTTGGGTGACCACCAGGA-3; 5′-AGCTAAGTATTAGAGCGGCGGCA-3′), Arginase-1 (5′-TGACATCAACACTCCCCTGACAAC-3′; 5′-GCCTTTTCTTCCTTCCCAGCAG-3′), Mannose-receptor-CD206 (5′-CATGAGGCTTCTCCTGCTTCTG-3; 5′-TTGCCGTCTGAACTGAGATGG-3), GAPDH (5′-ACGGCCGCATCTTCTTGTGCA-3′; 5′-CGGCCAAATCCGTTCACACCGA-3′).