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Recombinant c5a

Manufactured by Merck Group
Sourced in China, United States

Recombinant C5a is a laboratory reagent produced through recombinant DNA technology. It is a recombinant form of the human complement protein C5a, which plays a role in the immune system's inflammatory response. The core function of Recombinant C5a is to serve as a research tool for studying the biological functions and signaling pathways of the C5a protein.

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3 protocols using recombinant c5a

1

Endovascular Injury-Induced Neointima Formation

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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.
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2

Comprehensive Leukocyte Phenotyping Protocol

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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).
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3

Immune Profiling of Lepirudin-treated PBMCs

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PBMC were isolated from whole-blood anticoagulated with lepirudin (Refludan®, Celgene). Recombinant C5a, ultrapure cholesterol, 1-propanol were purchased from Sigma-Aldrich, and TNF from Genentech. The following reagents were used for qRT-PCR analyses: RNeasy Mini kit (Qiagen), DNase and High Capacity RNA-to-cDNA Kit (Applied Biosystems) and PerfeCTa® qPCR FastMix™ (Quanta Biosciences). Assays were from Applied Biosystems: GAPDH (Hs99999905_m1), NLRP3 (Hs00918082_m1), TNF (Hs00174128_m1), and IL-1β (Hs01555410_m1) and primers from SYBR as shown in Supplementary Table I. Antibodies used for immunohistochemistry: polyclonal rabbit anti-human C1q (A0136, Dako), normal rabbit IgG (Ab105-C, R&D systems), goat anti-mouse conjugated to Alexa 647 (Invitrogen), anti-C5b-9 (DIA-011, Antibodyshop), anti-CD68 (Dako Agilent M0814), anti-CD20 (Dako Agilent M0755), anti-CD3 (Dako Agilent A0452), anti-CD15 (BD Pharmingen 559045), control IgG2a (BD Pharmigen). Antibodies used for flow cytometry included anti-CD14 FITC (M5E2), anti-CD4 PerCP-CY 5.5 (RPA-T4), anti-CD8 BV605 (SK1) and anti-CD19 BV711 (SJ25C1) and were purchased from BD Pharmigen, and anti-CD3 APC (UCHT1) were from Invitrogen. Fluorescence-minus-one (FMO) was used as control.
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