Trem 1 sirna

Lipofectamine® 2000 Transfection Reagent and TREM-1 siRNA were purchased from Invitrogen (Carlsbad, USA). TRIzol, LPS, and human LDL were purchased from Sigma-Aldrich (St. Louis, USA). LP17 (LQVTDSGLYRCVIYHPP) and a sequence-scrambled control-peptide (TDSRCVIGLYHPPLQVY) were produced by GL Biochem (Shanghai) LTD, as described by Gibot et al. [18 (link)]. The peptides were obtained with good yields (>99%), purified, and confirmed by preparative chromatography. These peptides were free of endotoxin. The anti-CD14 and CD4 magnetic beads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The ELISA kits, CD1a-FITC, CD40-FITC, CD86-FITC, CD83-FITC, CD14-FITC, and HLA-DR-PE antibodies were purchased from BD (Franklin Lakes, USA). The anti-TREM-1 primary antibody and Human sTREM-1 ELISA kit were purchased from R&D (HK, China). The anti-SOCS1and GAPDH primary antibodies were purchased from Cell Signaling (Denver, USA). The cDNA Synthesis Kit and Premix Ex Taq SYBR Green PCR Kit were purchased from Takara (Shiga, Japan). Recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) were purchased from R&D Systems (Minneapolis, USA).

Murine macrophage cells (RAW264.7), purchased from Shanghai Bogoo Biotechnology Company (Shanghai, China), were routinely maintained in RPMI 1640 media (containing 11.1 mM glucose) supplemented with 10% fetal bovine serum (Sciencell, USA) and incubated at 37°C in 5% CO2. Firstly, RAW264.7 cells were stimulated with 25 mM high glucose for 24 h. Second, in order to examine the effect of TREM-1 on high-glucose induced macrophage polarization, the RAW264.7 cells were treated with TREM-1 siRNA (Invitrogen, America) and the cells were washed three times with PBS followed by RNA harvest for quantitative real-time polymerase chain reactions (RT-PCR) and the proteins for western blotting.