The expressions of the M1 marker iNOS and the M2 marker CD163 were evaluated by immunofluorescent staining assays to evaluate the polarization of macrophages. iNOS and CD163 were labeled with rabbit anti-mouse iNOS (1:250, Abcam, USA), mouse anti-mouse CD163 (1:250, Bioss, China), and donkey anti-rabbit Alexa Fluor 555 (1:250, Bioss, China) and simultaneously stained with mouse anti-mouse CD163 (1:250, Bioss, China). The analysis of immunofluorescence intensity was performed as previously described.
The relative mRNA expression of anti-inflammatory cytokines was also detected by PCR with the abovementioned method. The sequences of the primers of IL-18, CD206, IL-10 and iNOS are provided in Table S1.
TNF-α, NF-kB, and IL-10, which are inflammatory-associated cytokines, were detected by enzyme-linked immunosorbent assay kits (Cusabio, Wuhan, China). After culture for 3 days as previously described, the supernatants of macrophages were collected after centrifugation at 1000 g for 10 min. Concentrations of TNF-α, NF-kB, IL-10 in the supernatant were quantified using an enzyme-labeled instrument (Epoch; BioTek Instruments, Inc., Winooski, VT, USA) according to the manufacturer’s instructions.
The relative mRNA expression of anti-inflammatory cytokines was also detected by PCR with the abovementioned method. The sequences of the primers of IL-18, CD206, IL-10 and iNOS are provided in Table S1.
TNF-α, NF-kB, and IL-10, which are inflammatory-associated cytokines, were detected by enzyme-linked immunosorbent assay kits (Cusabio, Wuhan, China). After culture for 3 days as previously described, the supernatants of macrophages were collected after centrifugation at 1000 g for 10 min. Concentrations of TNF-α, NF-kB, IL-10 in the supernatant were quantified using an enzyme-labeled instrument (Epoch; BioTek Instruments, Inc., Winooski, VT, USA) according to the manufacturer’s instructions.