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      Purified Rat Anti-Human GM-CSF
      Purified Rat Anti-Human GM-CSF
      Expression of GM-CSF by stimulated human peripheral blood mononuclear cells (PBMC). Ficoll™-separated human PBMC were stimulated with Purified NA/LE Mouse Anti-Human CD3 (1 µg/ml final concentration; Cat. No. 555329), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL-4 (10 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL-4 for 3 days. Finally, the cells were harvested and stimulated for 6 hours with PMA (50 ng/ml final concentration;  Sigma, Cat. #P-8139) and calcium ionophore A23187 (250 ng/ml final concentration; Sigma, Cat. #C-9275) in the presence of  BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were harvested, stained with PE-Cy™5 Mouse Anti-Human CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.25 µg of PE Rat anti-Human GM-CSF (Cat. No. 554507) antibody by using the intracellular staining protocol (see Left panel). To demonstrate specificity of staining, the binding of PE Rat Anti-Human GM-CSF was blocked by preincubation of the antibody conjugate with recombinant human GM-CSF (0.1 µg; Cat. No. 550068; see Center panel) and by preincubation of the fixed/permeabilized cells with Purified Rat Anti-Human GM-CSF (5 µg; Cat. No. 554503; see Right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on the autofluorescence control and verified using the ligand-blocking and unlabeled antibody blocking controls.
      Purified Rat Anti-Human GM-CSF
      Expression of GM-CSF by stimulated human peripheral blood mononuclear cells (PBMC). Ficoll™-separated human PBMC were stimulated with Purified NA/LE Mouse Anti-Human CD3 (1 µg/ml final concentration; Cat. No. 555329), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL-4 (10 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL-4 for 3 days. Finally, the cells were harvested and stimulated for 6 hours with PMA (50 ng/ml final concentration;  Sigma, Cat. #P-8139) and calcium ionophore A23187 (250 ng/ml final concentration; Sigma, Cat. #C-9275) in the presence of  BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were harvested, stained with PE-Cy™5 Mouse Anti-Human CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.25 µg of PE Rat anti-Human GM-CSF (Cat. No. 554507) antibody by using the intracellular staining protocol (see Left panel). To demonstrate specificity of staining, the binding of PE Rat Anti-Human GM-CSF was blocked by preincubation of the antibody conjugate with recombinant human GM-CSF (0.1 µg; Cat. No. 550068; see Center panel) and by preincubation of the fixed/permeabilized cells with Purified Rat Anti-Human GM-CSF (5 µg; Cat. No. 554503; see Right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on the autofluorescence control and verified using the ligand-blocking and unlabeled antibody blocking controls.
      Product Details
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      BD Pharmingen™
      CSF2; Colony stimulating factor 2 (granulocyte-macrophage); CSF; GMCSF
      Human (QC Testing)
      Rat LEW, also known as Lewis IgG2a
      Recombinant human GM-CSF
      Intracellular block/flow cytometry (Tested During Development), Immunoprecipitation/Western blot (Reported)
      0.5 mg/ml
      AB_398564
      Aqueous buffered solution containing ≤0.09% sodium azide.
      RUO


      Preparation And Storage

      Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
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      Recommended Assay Procedures

      Blocking Control for Intracellular Staining: The purified BVD2-21C11 antibody (Cat. No. 554503) can be used as a blocking control to demonstrate specificity of human GM-CSF staining by the PE-BVD2-21C11 (Cat. No. 554507). To perform this control, the fixed/permeabilized cells (~ 1 million) can be incubated with 1 - 10 µg of unlabeled BVD2-21C11 antibody (Cat. No. 554503) for 20 minutes at 4°C, prior to staining with PE-BVD2-21C11 antibody (eg, 0.1 - 0.5 µg mAb/ 1 million cells). The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.  For specific methodology, please visit the protocols section under "Cytokines (Intracellular Staining)" at our website: http://www.bdbiosciences.com/us/s/resources.

      ELISA Detection: The biotinylated BVD2-21C11 antibody, Cat. No. 554505, is useful as a detection antibody for a sandwich ELISA for measuring human GM-CSF protein levels.  Biotinylated BVD2-21C11 antibody can be paired with the purified BVD2-23B6 antibody (Cat. No. 554502) as the capture antibody, with recombinant human GM-CSF (Cat. No. 550068) as the standard. For specific methodology please visit the protocols sections or the chapter on ELISA in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com. This ELISA pair is recommended primarily for measuring cytokine from experimental cell culture systems. Note: For testing GM-CSF in serum or plasma, our BD OptEIA™ ELISA Set (Cat. No. 555126) is recommended.

      IP: The purified BVD2-21C11 antibody has been reported to be useful for immunoprecipitation studies.  Please note that this application is not routinely tested at BD Biosciences Pharmingen.

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      Product Notices

      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. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
      5. Cy is a trademark of GE Healthcare.
      6. 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.
      7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      554503 Rev. 3
      Antibody Details
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      BVD2-21C11

      The BVD2-21C11 monoclonal antibody specifically binds to human Granulocyte/Macrophage - Colony Stimulating Factor (GM-CSF). Human GM-CSF is encoded by the CSF2 gene and is also known as Colony Stimulating Factor 2.  GM-CSF is produced by activated T lymphocytes, macrophages, endothelial cells, fibroblasts, stromal cells and other cell types including B lymphocytes, mast cells, eosinophils, and osteoblasts.  GM-CSF stimulates the survival, proliferation and/or differentiation of various cell types including neutrophils, eosinophils, macrophages, dendritic cells, megakaryocytes, erythroid cells, endothelial cells and their precursors. The immunogen used to generate the BVD2-21C11 hybridoma was recombinant human GM-CSF. The BVD2-21C11 antibody has been reported to crossreact with GM-CSF from the rhesus monkey. BVD2-21C11 is a neutralizing antibody.

      554503 Rev. 3
      Format Details
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      Purified
      Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
      Purified
      554503 Rev.3
      Citations & References
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      View product citations for antibody "554503" on CiteAb

      Development References (5)

      1. Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
      2. Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific: ELISA, Immunoprecipitation). View Reference
      3. Bacchetta R, de Waal Malefijt R, Yssel H. Host-reactive CD4+ and CD8+ T cell clones isolated from a human chimera produce IL-5, IL-2, IFN-gamma and granulocyte/macrophage-colony-stimulating factor but not IL-4. J Immunol. 1990; 144(3):902-908. (Clone-specific: ELISA). View Reference
      4. Kita H, Ohnishi T, Okubo Y, Weiler D, Abrams JS, Gleich GJ. Granulocyte/macrophage colony-stimulating factor and interleukin 3 release from human peripheral blood eosinophils and neutrophils. J Exp Med. 1991; 174(3):745-748. (Clone-specific: ELISA). View Reference
      5. Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 1995; 188(1):117-128. (Methodology: IC/FCM Block). View Reference
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      554503 Rev. 3

       

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      Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


      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.