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      PE Rat Anti-Human GM-CSF
      PE 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™7 Mouse Anti-Human CD4 (Cat. No. 557852), 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.
      PE 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™7 Mouse Anti-Human CD4 (Cat. No. 557852), 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 staining (flow cytometry) (Routinely Tested)
      0.2 mg/ml
      AB_395440
      Aqueous buffered solution containing ≤0.09% sodium azide.
      RUO


      Preparation And Storage

      Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.
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      Recommended Assay Procedures

      Immunofluorescent Staining and Flow Cytometric Analysis: The BVD2-21C11 antibody is useful for the immunofluorescent staining and flow cytometric analysis to identify and enumerate GM-CSF producing cells within mixed cell populations. (see Figure, Left panel ). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤0.5 µg mAb/million cells). For specific methodology, please visit our web site, http://www.bdbiosciences.com/us/s/resources, and go to the protocols section under "Cytokines (Intracelullar Staining)".

      A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the fluorochrome-conjugated BVD2-21C11 antibody with ligand (e.g., recombinant human GM-CSF, Cat No. 550068) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled BVD2- 21C11 antibody (Cat. No. 554503) prior to staining. The intracellular staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.  A suitable rat IgG2a isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponinpermeabilized mouse or human cells is PE-R35-95 immunoglobulin (Cat. No. 554689); use at comparable concentrations to antibody of interest (e..g., ≤0.5 µg mAb/1 million cells).

      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.  For testing GM-CSF in serum or plasma, the OptEIA™ ELISA Set (Cat. No. 555126) is recommended.

      IP/WB: The purified BVD2-21C11 antibody has been reported to be useful for immunoprecipitation studies and for Western blotting.  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      5. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
      6. Cy is a trademark of GE Healthcare.
      7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      554507 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.

      554507 Rev. 3
      Format Details
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      PE
      R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
      altImg
      PE
      Yellow-Green 488 nm, 532 nm, 561 nm
      496 nm, 566 nm
      576 nm
      554507 Rev.3
      Citations & References
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      View product citations for antibody "554507" 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, Western blot). 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: Flow cytometry, IC/FCM Block). View Reference
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      554507 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.