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      PE Mouse Anti-Human LT-α
      PE Mouse Anti-Human LT-α
      Detection of LT-α expression by stimulated  human lymphocytes using PE-359-81-11. Human PBMC  were stimulated with soluble anti-human CD3 mAb (1 µg/ml, clone UCHT1, Cat. No. 555329) and recombinant human IL-2 (10 ng/ml, Cat. No. 554603) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hr with immobilized anti-human CD3 mAb (10 µg/ml) and soluble anti-human CD28 (20 ng/ml, clone CD28.2, at. No. 555725) in the presence of 2 µM  GolgiStop™ (aka, monensin; Cat. No. 554724). The cells were harvested, stained with PE-Cy5™-anti-CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.25 µg of PE- anti-human LT-α antibody by using the BD Pharmingen™ staining protocol (left panel). To demonstrate specificity of staining, the binding by PE-359-81-11 was blocked by each of the following: 1) preincubation of the conjugated antibody with excess  recombinant human LT-α (Cat. No. 554619; right panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled 359-81-11 mAb (data not shown) prior to staining with the PE-  359-81-11. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
      PE Mouse Anti-Human LT-α
      Detection of LT-α expression by stimulated  human lymphocytes using PE-359-81-11. Human PBMC  were stimulated with soluble anti-human CD3 mAb (1 µg/ml, clone UCHT1, Cat. No. 555329) and recombinant human IL-2 (10 ng/ml, Cat. No. 554603) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hr with immobilized anti-human CD3 mAb (10 µg/ml) and soluble anti-human CD28 (20 ng/ml, clone CD28.2, at. No. 555725) in the presence of 2 µM  GolgiStop™ (aka, monensin; Cat. No. 554724). The cells were harvested, stained with PE-Cy5™-anti-CD4 (Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.25 µg of PE- anti-human LT-α antibody by using the BD Pharmingen™ staining protocol (left panel). To demonstrate specificity of staining, the binding by PE-359-81-11 was blocked by each of the following: 1) preincubation of the conjugated antibody with excess  recombinant human LT-α (Cat. No. 554619; right panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled 359-81-11 mAb (data not shown) prior to staining with the PE-  359-81-11. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
      Product Details
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      BD Pharmingen™
      TNF-β
      Human (QC Testing)
      Mouse IgG1
      Recombinant Human LT-α (TNF-β)
      Intracellular staining (flow cytometry) (Routinely Tested)
      0.2 mg/ml
      4049
      AB_395478
      Aqueous buffered solution containing ≤0.09% sodium azide.
      RUO


      Preparation And Storage

      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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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      Recommended Assay Procedures

      Immunofluorescent Staining and Flow Cytometric Analysis: The 359-81-11 antibody is useful for intracellular immunofluorescent staining and flow cytometric analysis to identify and enumerate LT-α producing cells within mixed cell populations. PE-conjugated 359-81-11 antibody is

      especially suitable for these studies. The use of a specificity control,  such as one of the following, is suggested: 1) recombinant human TNF-β (Cat No. 554619), 2) unlabeled 359-81-11 antibody (Cat. No. 554554), or 3) mouse IgG1 isotype control, PE-MOPC-21 (Cat. No. 554680).

      ELISA Detection: The biotinylated 359-81-11 antibody is useful as a detection antibody for a sandwich ELISA for measuring human LT-α  protein levels in cell culture supernatants.  Biotinylated 359-81-11 antibody can be paired with the purified 359-238-8 antibody (Cat. No. 554557) as the capture antibody, with recombinant LT-α (Cat. No. 554619) as the standard. For specific methodology, please visit the protocols section or chapter on ELISA in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.

      Neutralization: The NA/LE™ 359-81-11 antibody has been reported to be useful for neutralization of human LT-α bioactivity.   A suitable NA/LE mouse IgG1 isotype control to match the 359-81-11 antibody is the 107.3 antibody, (Cat. No. 554721).

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      Product Notices

      1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
      2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      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.
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      554556 Rev. 1
      Antibody Details
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      359-81-11

      The 359-81-11 antibody reacts with human Lymphotoxin-α  (LT-α, also known as tumor necrosis factor-β or TNF-β).   The immunogen used to generate the 359-81-11 hybridoma was recombinant human LT-α.  This is a neutralizing antibody.  

      554556 Rev. 1
      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
      554556 Rev.1
      Citations & References
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      Development References (3)

      1. Meager A, Parti S, Leung H, Peil E, Mahon B. Preparation and characterization of monoclonal antibodies directed against antigenic determinants of recombinant human tumour necrosis factor (rTNF). Hybridoma. 1987; 6(3):305-312. (Clone-specific: ELISA, Neutralization). View Reference
      2. Meager A, Parti S, Leung H, et al. A two-site sandwich immunoradiometric assay of human lymphotoxin with monoclonal antibodies and its applications. J Immunol Methods. 1987; 104(2):31-42. (Clone-specific: ELISA, Neutralization). View Reference
      3. 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
      554556 Rev. 1

      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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