Preparation And Storage
Recommended Assay Procedures
Immunofluorescent Staining and Flow Cytometric Analysis: Purified Mouse Anti-Human CD120a (Cat. No. 550514) can be used for the immunofluorescent staining (≤ 1 µg antibody/10e6 cells) and flow cytometric analysis of normal human lymphoid cells or cell lines to measure their expressed levels of surface TNFRI. An appropriate purified isotype control antibody is Purified Mouse IgG2a, κ Isotype Control (Cat. No. 555571). A three-step staining protocol is recommended for maximizing the detection of TNFRI expressed by cells as detailed in the figure legend (see below). As a demonstration of specificity, the binding of MABTNFR1-B1 to TNFRI is inhibited when human TNFRI+ target cells are preincubated with saturating levels of recombinant human TNF at 4°C, i.e., when the TNFRI are bound with ligand. Based on our data, recombinant human TNF at ≥ 50 ng/10e6 cells is sufficient to completely inhibit the binding of MABTNFR1-B1 (0.06 µg/10e6 cells). Please note that as a consequence of in vivo or in vitro activation, cell surface TNFRI can either be shed by cells or transiently expressed at higher levels. As a result, cellular activation can affect the cell's overall expressed level of surface TNFRI.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Ficoll-Paque is a trademark of Amersham Biosciences Limited.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
The TNFR1-B1 monoclonal antibody specifically recognizes the extracellular domain of CD120a. CD120a is encoded by TNFRSF1A (Tumor necrosis factor receptor superfamily member 1A). CD120a is also known as the 55 kDa receptor for the human cytokines, tumor necrosis factor (TNF) and lymphotoxin-alpha (LT-a). This receptor is referred to as the p55 or Type I Tumor Necrosis Factor Receptor (TNFRI). TNFRI are expressed by a variety of cell lines, tumor cells, and normal cell types including T cells, monocytes, macrophages, neutrophils, endothelial cells, hepatocytes, chondrocytes, and fibroblasts. Naive B cells express very low or undetectable levels of TNFRI whereas mature erythrocytes and platelets are uniformly negative for TNFRI expression. MABTNFR1-B1 specifically binds to natural and recombinant truncated forms of TNFRI.
Development References (10)
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Erikstein BK, Smeland EB, Blomhoff HK, et al. Independent regulation of 55-kDa and 75-kDa tumor necrosis factor receptors during activation of human peripheral blood B lymphocytes. Eur J Immunol. 1991; 21(4):1033-1037. (Biology). View Reference
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Hohmann HP, Remy R, Brockhaus M, van Loon AP. Two different cell types have different major receptors for human tumor necrosis factor (TNF alpha). J Biol Chem. 1989; 264(25):14927-14934. (Biology). View Reference
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Zola H. Detection of cytokine receptors by flow cytometry. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, ed. Current Protocols in Immunology. New York: Green Publishing Associates and Wiley-Interscience; 1995:6.21.1-6.21.18.
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.