Recombinant c5a

Animal care and protocol for this study were in accordance with the Animal Experiment Guidelines of Harbin Medical University (Harbin, China) and ethical approval was obtained from Harbin Medical University. C57BL/6N, 16-week-old mice (Fuzhong Biotechnology, Shanghai, China) were subjected to transluminal endovascular wire injury of the bilateral femoral artery, as reported previously (8 (link)). All the mice were fed normal chow and water ad libitum and kept under normal diurnal cycle at room temperature (22°C). Over a course of two weeks, recombinant C5a (Sigma, Shanghai, China; 1 μg/25 g of body weight) and recombinant C4a (1 μg/25 g of body weight) were administered via an Alzet mini-osmotic pump (American Health & Medical Supply International Corp, Chengdu, Sichuan, China) into the subcutaneous perifemoral artery following endovascular injury, according to a previously described method (16 (link)). Image analysis software was used to measure the neointima and media of the femoral artery. The relative mRNA expression levels of CD68 and F4/80 in femoral arterial tissue were assessed by quantitative polymerase chain reaction (qPCR), and the protein levels of TNF-α, IL-6 and MCP-1 in femoral arterial tissue were assessed by western blot analysis.

Anti-CD14 [PE mouse anti-human, clone M5E2], anti-CD16 [PE mouse anti-human, clone 3G8], anti-CCR1 [Alexa Fluor 647 mouse anti-human CD191, clone 53504], anti-CCR2 [Alexa Fluor 647 mouse anti-human CD192, clone 48607], κ Isotype Control Alexa Fluor 647 [mouse IgG2b, clone 27–35], anti-BLT1 [mouse anti-human, clone 14F11], anti-FPR1 [mouse anti-human, clone 556015], anti-C5aR [mouse anti-human, clone D53–1472] and polyclonal PE goat anti-mouse were purchased from BD Biosciences (Temse, Belgium). Anti-CCR5 [mouse anti-human, clone 45531] was purchased from R&D Systems (Abingdon, UK). Anti-CD15 [FITC mouse anti-human, clone HI98] and anti-CD62L (L-selectin) [APC mouse anti-human, clone DREG-56] were obtained from eBioscience (Vienna, Austria). Anti-CD14 [APC mouse anti-human, clone M5E2] and APC anti-mouse IgG2a isotype control [clone MOPC-173] were obtained from Biolegend (San Diego, USA). LTB4 and recombinant human CCL2 were bought from Peprotech (Rocky Hill, NY), fMLP and recombinant C5a from Sigma (St. Louis, USA).