The monoclonal antibody 20/70 recognizes the mouse receptor for C5a (CD88). The mouse anaphylatoxin complement factor C5a, a split product of C5, is one of the most potent inflammatory chemoattractants and interacts with two C51 receptors, C5aR and C5L2. Most of the C5a effects are mediated through C5aR. C5aR is known to be crucial in the initiation of acute inflammatory responses. C5a receptor is a seven transmembrane GTP-binding-protein-coupled receptor that belongs to the rhodopsin supergene family. The 45 kDa C5a receptor is expressed by leukocytes such as neutrophils, eosinophils, basophils, monocytes, dendritic cells and mast cells. C5aR can be expressed on both immune and nonimmune cells. Nonimmune cells expressing C5aR include vascular endothelial cells, cardiomyocytes and bronchial epithelial cells. Signaling of C5aR involves intracellular calcium mobilization and activation of among others protein kinase- B (PKB) and protein phospholipase D (PLD) pathways. High C5aR expression levels have been associated with the pathogenesis of many inflammatory conditions, autoimmune and neurodegenerative diseases like rheumatoid arthritis, multiple sclerosis, atherosclerosis and inflammatory bowel diseases. In vivo blockade of C5aR greatly reduces inflammatory injury. The monoclonal antibody 20/70 has been used in functional studies and is capable to prevent the binding of C5a to its receptor.
Flow cytometry, Functional studies, Immuno fluorescence
FC: 2x10^5 cells were blocked with human and murine IgG on ice for 20’. Cells were stained with 10μg antibody 20/70 for 45’ in PBS/1.5S. For analysis with FACS cells were washed with PBS/1%formaldehyde (Ref.2).
FS: Before migration towards C5a was induced, 2x105 cells were incubated for 30’ with 10μg antibody 20/70 in a transwell migration assay (Ref.2).
IF: Cells were cultured on coverslips, fixed with 4% PFA for 20’, blocked with 3% BSA for 2h and stained with antibody 20/70 for 2h (Ref.6).
Dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50. For functional studies, in vitro dilutions have to be optimized in user’s experimental setting.