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      PE Mouse Anti-Human IL-8
      PE Mouse Anti-Human IL-8
      Expression of IL-8 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (10 ng/ml final concentration) in the presence of 2 µM GolgiStop™ (Cat. No. 554724). The PBMC were harvested, stained with FITC Mouse Anti-Human CD14 (Cat. No. 555397), fixed, permeabilized, and subsequently stained with 0.25 µg of PE Mouse Anti-Human IL-8 (Cat. No. 554720) following Pharmingen's staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, the binding of PE Mouse Anti-Human IL-8 was blocked by the preincubation of the conjugated antibody with recombinant human IL-8 (0.25 µg, Cat. No. 554609; center panel), and by preincubation of the fixed/permeabilized cells with Purified Mouse Anti-Human IL-8 (2.5 µg, Cat. No. 554717/550419; right panel) prior to staining with the PE Mouse Anti-Human IL-8. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (center) and unlabelled antibody (right) blocking specificity controls.
      PE Mouse Anti-Human IL-8
      Expression of IL-8 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (10 ng/ml final concentration) in the presence of 2 µM GolgiStop™ (Cat. No. 554724). The PBMC were harvested, stained with FITC Mouse Anti-Human CD14 (Cat. No. 555397), fixed, permeabilized, and subsequently stained with 0.25 µg of PE Mouse Anti-Human IL-8 (Cat. No. 554720) following Pharmingen's staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, the binding of PE Mouse Anti-Human IL-8 was blocked by the preincubation of the conjugated antibody with recombinant human IL-8 (0.25 µg, Cat. No. 554609; center panel), and by preincubation of the fixed/permeabilized cells with Purified Mouse Anti-Human IL-8 (2.5 µg, Cat. No. 554717/550419; right panel) prior to staining with the PE Mouse Anti-Human IL-8. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (center) and unlabelled antibody (right) blocking specificity controls.
      Product Details
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      BD Pharmingen™
      IL8; CXCL8; GCP-1; LYNAP; MDNCF; MONAP; NAP-1; emoctakin
      Human (QC Testing)
      Mouse IgG2b
      Recombinant Human IL-8
      Intracellular staining (flow cytometry) (Routinely Tested)
      0.2 mg/ml
      AB_395529
      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 Cytometry: The PE Mouse Anti-Human IL-8 (Cat. No. 554720) can be used for multicolor immunofluorescent staining and flow cytometric analyses to identify and enumerate IL-8 producing cells within mixed cell populations (see figure). 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 protocols under "Intracellular Flow" at our website: http://www.bdbiosciences.com/us/s/resources/.

      A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated G265-8 antibody with ligand (e.g., recombinant human IL-8; Cat. No. 554609) prior to staining, or 2) pre-block the fixed/ permeabilized cells with Purified Mouse Anti-Human IL-8 (Cat. No. 554717/550419) prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. An appropriate PE-mouse IgG2b isotype control to use on fixed and permeabilized cells is PE Mouse IgG2b, κ Isotype Control (Cat. No. 555058).

      Cytokine ICC: The G265-8 antibody is useful for immunocytochemical staining. Purified Mouse Anti-Human IL-8 (Cat. No. 550419/554717) is tested in the ICC application.

      ELISA Detection: The Biotin Mouse Anti-Human IL-8 antibody (Catalog No. 554718) is useful as a detection antibody in a sandwich ELISA for measuring human IL-8 protein levels. Biotin Mouse Anti-Human IL-8 antibody can be paired with the Purified Mouse Anti-Human IL-8 (Cat. No. 554716) with recombinant human IL-8 (Cat. No. 554609) as the standard. This ELISA pair is recommended primarily for measuring cytokine from experimental cell culture systems. For detection of IL-8 in serum or plasma, the Human IL-8 BD OptEIA™ ELISA Set (Cat. No. 555244) or OptEIA™ ELISA Kit (Cat. No. 550999) is recommended.

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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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      554720 Rev. 3
      Antibody Details
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      G265-8

      The G265-8 monoclonal antibody specifically binds to both the 72 and 77 amino acid isoforms of human Interleukin-8 (IL-8). IL-8 is secreted as an 8-9 kDa, non-glycosylated proinflammatory chemokine protein also known as chemokine (C-X-C motif) ligand 8 (CXCL8). IL-8 is synthesized as a 99 amino acid precursor that is proteolytically processed into several isoforms. The 72 amino acid isoform is produced by monocytes, macrophages, granulocytes, epithelial cells, and fibroblasts in response to pro-inflammatory stimuli including cytokines and microbial agents. It is also expressed by endothelial cells, fibroblasts, keratinocytes, lymphocytes, and a variety of tumor cells. In response to IL-4, IL-10 and TGFβ, the cellular production of IL-8 is inhibited. IL-8 is crucial for the activation and recruitment of neutrophils to inflammatory sites. IL-8 is also a chemoattractant for basophils and T-lymphocytes. IL-8 possesses angiogenic activity and can be associated with tumor angiogenesis and metastasis. The 77 amino acid IL-8 isoform is primarily produced by endothelial cells. This larger isoform is reportedly a less potent neutrophil activator than the 72 amino acid isoform. IL-8 binds to and signals through two G-protein-coupled receptors, IL-8RA (CXCR1/CD181) and IL-8RB (CXCR2/CD182).

      554720 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
      554720 Rev.3
      Citations & References
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      Development References (2)

      1. Matsushima K, Oppenheim JJ. Interleukin 8 and MCAF: novel inflammatory cytokines inducible by IL 1 and TNF. Cytokine. 1989; 1(1):2-13. (Biology). View Reference
      2. 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). View Reference
      554720 Rev. 3

      Please refer to Support Documents for Quality Certificates


      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.

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