Apc conjugated anti mouse f4 80

For investigating the effect of FTCD on macrophage polarization in vitro, the supernatant of Hepa1-6 cells transfected with the pcDNA3.1(-) plasmid or pcDNA3.1-FTCD plasmid, as well as the supernatant of AML12 cells transfected with si-NC or si-FTCD was collected and used for the incubation of Raw264.7 cells for 48 h. Then, Raw264.7 cells were washed and resuspended with precooled PBS, and incubated with specific antibodies, PE-conjugated anti-mouse CD86 (11-0862-82, 0.125 μg/test) and FITC-conjugated anti-mouse CD206 (12-2061-82, 0.125 μg/test) from Thermo Fisher Scientific (Waltham, MA, USA), at 4 °C in the dark for 30 min. For detecting the effect of FTCD on macrophage infiltration in vivo, the liver was isolated from the intrahepatic injected mouse described above. Single-cell suspensions were made, and incubated with specific antibodies, including SB436-conjugated anti-mouse CD45 (62-0451-82, 0.5 μg/test), APC-conjugated anti-mouse F4/80 (17-801-82, 2 μg/test), PE-conjugated anti-mouse CD86 and FITC-conjugated anti-mouse CD206 from Thermo Fisher Scientific, at 4 °C in the dark for 30 min. Flow cytometry was performed according to standard procedures on a BD FACSArica III (Piscataway, NJ, USA).

Ginseng extract (Ginsenosides ≥80%, designated as R) and EGCG (designated as E) with purity higher that 94% were purchased from Shanghai Novanat Co., Ltd. (Shanghai, China). PDX (designated as D) with purity higher that 90% was purchased from Tate & Lyle Trading Co., Ltd. (Shanghai, China). The complex of selected nutrients was proposed based on the experimental design and designated as the combination of the abbreviation of the corresponding nutrient (ERD). Lipopolysaccharide (LPS) was purchased from Santa Cruz Biotechnology (California, United States). Cell Counting Kit-8 (CCK-8) reagent was purchased from Meilunbio (Dalian, China). Anti-mouse CD16/32, APC-conjugated anti-mouse F4/80, FITC-conjugated anti-mouse CD86, and PE-conjugated anti-mouse CD206 antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, United States) for flow cytometry analysis. Recombinant mouse IFN-γ and IL-4 were provided by Thermo Fisher Scientific. Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β analysis were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Neutral red was from sourced from Aladdin (Shanghai, China). An apoptosis kit was purchased from Thermo Fisher Scientific. All reagents used in this study were of analytical grade unless otherwise indicated.

Flow cytometry analysis was performed as previously described 3 using the following antibodies: APC‐conjugated antimouse F4/80 (eBioscience, San Diego, CA, USA), APC‐conjugated antimouse CD16/32 (Biolegend, San Diego, CA, USA), APC‐conjugated antimouse CD206 (Biolegend), PE‐conjugated antimouse Dectin‐1 (Biolegend), PE‐conjugated antimouse CD4 (eBioscience) and FITC‐conjugated antimouse CD69 (eBioscience).