Optimizing TREM-1 Silencing in Macrophages

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).