mouse anti human c5a antibody  (R&D Systems)

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: The co-expression of C5AR1 and its ligand C5a in intracranial aneurysm lesions. Specimens of intracranial aneurysm (IA) lesions (Aneurysm) or control arterial walls (Superficial temporal artery in ( A ), Anterior cerebral artery-olfactory artery bifurcation in ( B )) from human cases ( A ) or rats ( B ) were harvested and examined in immunohistochemistry. The representative images of immunofluorescent staining of IA lesions for C5AR1 (green), C5a/C5a des-arg (red in ( A )), C5 (red in ( B )), and merged images with nuclear staining by DAPI (blue) are shown. A demagnified image of lower panels in ( B ) is shown on the right. The square in the demagnified image corresponds to the area shown in the magnified images. Scale bar: 50 μm.

Article Snippet: The incubation mixture was then subjected to a western blot analysis using mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech).

Techniques: Expressing, Control, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: Tissue-type Plasminogen Activator as an up-stream factor to produce C5a from C5. ( A ) The expression of tissue-type Plasminogen Activator (tPA) and Plasminogen in intracranial aneurysm (IA) lesions. IA lesions from human cases or induced in rats were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for tPA (green), Plasminogen (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm. ( B ) Co-expression of tPA with C5a in IA lesions. Human IA lesions were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for C5a/C5a des-arg which is the derivative of C5a (green), tPA (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm. ( C ) Enzymatic cleavage of C5 into C5a by Plasmin. Recombinant C5 (100 μg/ml) was co-incubated with each dose of recombinant Plasmin as indicated (0–100 μg/ml) for 1.5 h in a cell-free system. The incubation mixture was subjected to western blot analyses. The representative images from the western blot analyses to detect C5 or C5a are shown. M; size marker. ( D ) The alternation in the coagulation-fibrinolysis system in IA lesions of rats. The heat map of genes related with the coagulation-fibrinolysis system from the comprehensive gene expression profile data set (GEO accession: GSE161044) is shown.

Article Snippet: The incubation mixture was then subjected to a western blot analysis using mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech).

Techniques: Expressing, Immunohistochemical staining, Staining, Recombinant, Incubation, Western Blot, Marker, Coagulation

Journal: Scientific Reports

Article Title: C5a–C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms

doi: 10.1038/s41598-024-53651-7

Figure Lengend Snippet: C5a–C5AR1 signaling cascade-dependent induction of pro-inflammatory factors. ( A – C ) The C5a–C5AR1 signaling cascade-dependent induction of pro-inflammatory genes in phorbol myristate acetate-primed cell line from human case with diffuse histiocytic lymphoma, U937 cell line. Cells were treated with recombinant C5a as indicated in the panels (0–10 h in ( A ), 0–100 nM in ( B ), 10 nM and 10 h in ( C ). Some cells were pre-treated with the selective C5AR1 inhibitor, W54011 (300 nM, 30 min) prior to the treatment with recombinant C5a. Total RNA was then purified from treated cells subjected to quantitative RT-PCR analyses to examine the induction of TNF (a gene for TNF-α), IL1B or PTGS2 (a gene for COX-2; cyclooxygenase-2). Data represents as mean ± s.e.m (( A ), n = 4; ( B , C ), n = 6). Statistical analysis was done by the Wilcoxon rank sum test in ( A ), the Kruskal–Wallis test followed by the Steel–Dwass test in ( B , C ). *p < 0.05. ( D ) Co-expression of C5a with pro-inflammatory factors in intracranial aneurysm (IA) lesions. IA lesions induced in rats were harvested subjected to immunohistochemical analyses. The images of immunofluorescent staining of IA lesions for C5ar1 (green), TNF-α (red), IL-1β (red), COX-2 (red), and merged images with nuclear staining by DAPI (blue) are shown. Scale bar: 50 μm.

Article Snippet: The incubation mixture was then subjected to a western blot analysis using mouse monoclonal anti-human C5a/C5a des-Arg antibody (clone 2952, #HM2079, Hycult Biotech).

Techniques: Recombinant, Purification, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Neuroscience

Article Title: Therapeutic Hypothermia Inhibits the Classical Complement Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy

doi: 10.3389/fnins.2021.616734

Figure Lengend Snippet: The Complement Cascade in HIE. Hypoxic insult induces expression of ischemia-induced “neoantigens” on the surface of vascular endothelial cells. These neoantigens are recognized by natural antibodies (IgM) initiating systemic classical complement pathway activation by binding C1q, characterized by a series of cleavages, culminating in the formation of a membrane attack complex (MAC) that has lytic activity. C5a and C3b are intermediates in the cascade, which execute chemotactic and phagocytic functions, respectively, while C3a has been shown to be anti-inflammatory in the acute phase. Components of the classical complement pathway (C1q, C3, and C9) are also endogenously produced in the brain, primarily in microglia after HI injury. C1q facilitates a non-inflammatory uptake of apoptotic neurons by microglia, thus limiting the exposure of surrounding neurons to toxic intracellular contents such as glutamate. Thus, microglial synthesis of C1q is a neuroprotective mechanism, and emphasizes the role of microglia and C1q in cellular homeostasis. However, in HI injuries, the extent of the damage amplifies the cascade, and mediators such as C5a perpetuate the inflammatory damage. HT (blue arrows) modulates the complement pathway in HIE by decreasing the microglial expression of C1q, C3, C9, systemic production of C5a, resulting in decreased neuronal apoptosis.

Article Snippet: Samples were probed with either 1:50 goat anti-human C1q (Complement Technology) in NDS or 1:200 mouse anti-human C5a (Human Complement Component C5a DuoSet ELISA, R&D Systems, Inc., Minneapolis, MN, United States) in NGS for 1 h at RT.

Techniques: Expressing, Immunopeptidomics, Activation Assay, Binding Assay, Membrane, Activity Assay, Produced

Journal: Frontiers in Neuroscience

Article Title: Therapeutic Hypothermia Inhibits the Classical Complement Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy

doi: 10.3389/fnins.2021.616734

Figure Lengend Snippet: HT decreases systemic and brain complement expression in neonatal HIE. Whole brain C1q levels in NT animals were significantly higher than HT treated animals at 8, 12, and 24 h after the initial insult (a) . There were no significant differences in the expression of whole brain C3aR (b) or C5aR (c) between NT and HT treated groups. C1q levels in the plasma of NT animals were significantly higher than HT treated animals at 8, 12, and 24 h after injury (d) . Systemic C3a (b) and C5a (c) levels in NT animals were significantly higher than HT treated animals at 12 and 24 after injury. The differences in C5a persisted out to 48 h. ( n = 7 animals per group, solid lines/white bars – NT; dotted lines/gray bars – HT; error bars denote ±SD, * denotes P < 0.05).

Article Snippet: Samples were probed with either 1:50 goat anti-human C1q (Complement Technology) in NDS or 1:200 mouse anti-human C5a (Human Complement Component C5a DuoSet ELISA, R&D Systems, Inc., Minneapolis, MN, United States) in NGS for 1 h at RT.

Techniques: Expressing, Clinical Proteomics