The cells were harvested by adding TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions, and total RNA was subsequently isolated. Genomic DNA contamination was removed by treating with DNaseI (Thermo Fisher Scientific). After that, the DNA-free total RNA was reverse-transcribed by the RevertAid First strand cDNA Synthesis Kit (Thermo Fisher Scientific). The transcription levels of cytokine genes and macrophage phenotype markers were determined using qRT-PCR (Bio-Rad Laboratories, Inc., Hercules, CA). The specific primers were designed as listed in S1 Table . Amplification was performed using the MasterCycler Real-Time PCR system (Realplex4, Eppendorf, Hamburg, Germany), and it consisted of a first denaturation step at 95°C for 5 min, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s and a melting curve step at 65°C–95°C. Relative mRNA expression was calculated using the comparative Ct method with the formula 2−ΔΔCt [16 (link)].
The levels of cytokines secreted from mBMDMs, including TNF-α, IFN-γ, TGF-β, IL-10, and IL-4, were determined in the culture supernatant using commercial sandwich ELISA kits according to the manufacturer’s instructions (eBioscience, Thermo Fisher Scientific). The cytokine concentrations were determined from the average of triplicates, and standard curves were generated with recombinant cytokines.
The levels of cytokines secreted from mBMDMs, including TNF-α, IFN-γ, TGF-β, IL-10, and IL-4, were determined in the culture supernatant using commercial sandwich ELISA kits according to the manufacturer’s instructions (eBioscience, Thermo Fisher Scientific). The cytokine concentrations were determined from the average of triplicates, and standard curves were generated with recombinant cytokines.