Description
The immunogen used to raise R3 was C1q-binding protein preparation as described. Human C1qRp is a 631 a.a. protein (~66.5 kD) protein that is highly expressed on monocytes/macrophages, neutrophil granulocytes but not on T and B lymphocytes. C1qRp binds C1q, the recognition subunit of the first component (C1) of the complement pathway, as well as MBL (Mannose-binding-lectin) and SPA (Pulmonary Surfactant Protein A). Human C1qRp is involved in the C1q-mediated enhancement of phagocytosis. R3 is suitable to detect C1qRp expression on cells of myeloid lineage by flow cytometry, C1qRp in cellular lysates by Western blotting or immunoprecipitation. Pretreatment of monocytes with R3 neutralizes C1q-mediated enhancement of phagocytosis. In addition, when immobilized, R3 mimics C1q- MBL- or SPA-mediated enhancement of phagocytosis as reported. CDw93 (C1qRp) has been reported to define a human stem cell population with hematopoietic and hepatic potential.
Immunofluorescent Staining and Flow Cytometric Analysis: The staining technique and blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable mouse IgM, κ isotype control (Cat. No. 555581) for assessing the level of background staining on human cells is recommended: use at comparable concentrations to antibody of interest (eg, ≤ 0.5 µg Ab/1 million cells) (see Figure). For specific methodology, please visit the protocols section under "Multicolor Flow Cytometry" at http://www.bdbiosciences.com/us/s/resources. Western Blotting and immunoprecipitation: When run under non-reducing conditions, Human CD93 migrates as a 100 kDa protein; due to high level of glycosylation CD93 migrates as 126 kDa under reducing conditions. The R3 antibody is suitable to detect CD93 by Western blotting and immunprecipitation as described; however, the antibody is not tested for this application at BD Biosciences Pharmingen. Reactivity of the antibody with the reduced protein is dramatically decreased. Modulation of monocyte phagocytic activity: Pretreatment of monocytes with R3 neutralizes CD93-mediated enhancement of phagocytosis. In addition, when immobilized, R3 mimics CD93- MBL- or SPA-mediated enhancement of phagocytosis; however, the antibody is not tested for this application at BD Biosciences Pharmingen.
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. An isotype control should be used at the same concentration as the antibody of interest.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
4. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
