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PE Mouse Anti-Human MIP-1β
PE Mouse Anti-Human MIP-1β

Expression of MIP-1β by stimulated human monocytes. Human PBMC were stimulated with human IFN-γ (20 ng/ml final concentration; Cat. No. 554616/554617) for one hour followed by overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. No. L-8272) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, fixed, permeabilized, and stained with 0.03 µg of PE Mouse Anti-Human MIP-1β (Cat. 550078/561120; Left Panel) following Pharmingen's staining protocol. The data reflects gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, binding by the PE Mouse Anti-Human MIP-1β antibody was blocked by preincubation with recombinant human MIP-1β (0.25 µg; Middle panel) and by preincubation of the fixed/permeabilized cells with excess unlabeled D21- 1351 antibody (5 µg; custom order; Right panel) prior to staining with PE Mouse Anti-Human MIP-1β. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified using the unlabeled antibody blocking control.

Expression of MIP-1β by stimulated human monocytes. Human PBMC were stimulated with human IFN-γ (20 ng/ml final concentration; Cat. No. 554616/554617) for one hour followed by overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. No. L-8272) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, fixed, permeabilized, and stained with 0.03 µg of PE Mouse Anti-Human MIP-1β (Cat. 550078/561120; Left Panel) following Pharmingen's staining protocol. The data reflects gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, binding by the PE Mouse Anti-Human MIP-1β antibody was blocked by preincubation with recombinant human MIP-1β (0.25 µg; Middle panel) and by preincubation of the fixed/permeabilized cells with excess unlabeled D21- 1351 antibody (5 µg; custom order; Right panel) prior to staining with PE Mouse Anti-Human MIP-1β. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified using the unlabeled antibody blocking control.

Product Details
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BD Pharmingen™
Macrophage inflammatory protein 1-beta; CCL4; C-C motif chemokine 4; LAG-1
Human (QC Testing)
Mouse IgG1, κ
Recombinant Human MIP-1β
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_393549
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.

Recommended Assay Procedures

IF/Flow: For immunofluorescent staining and flow cytometric analysis, the D21-1351 antibody has been found useful to identify and enumerate MIP-1β producing cells within mixed cell populations. PE Mouse Anti-Human MIP-1β (Cat. No. 550078/561120) is especially suitable for these studies.

A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponinpermeabilized human cells is PE Mouse IgG1, κ Isotype Control (Cat. No. 554680); use at comparable concentrations to antibody of interest (e.g., . 0.5 µg mAb/1 million cells). A useful control for demonstrating specificity of staining is to pre-block the fixed/permeabilized cells with unlabeled D21-1351 antibody (custom order), prior to staining. The intracellular staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.

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.
550078 Rev. 2
Antibody Details
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D21-1351

The D21-1351 monoclonal antibody specifically binds to the human CC chemokine, MIP-1β (macrophage inflammatory protein-1β). Human MIP-1β shares approximately 75% homology with mouse MIP-1β at the amino acid level.  Expression of MIP-1β in human peripheral blood cells is induced by proinflammatory and mitogenic stimuli.  MIP-1β  is a chemoattractant for monocytes and lymphocytes. Human MIP-1β binds to receptors, CCR5 and CCR8. The human MIP-1β gene has been mapped to chromosome 17q11. The immunogen used to generate D21-1351 hybridoma was recombinant human MIP-1β.

550078 Rev. 2
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
550078 Rev.2
Citations & References
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Development References (9)

  1. Bernardini G, Hedrick J, Sozzani S. Identification of the CC chemokines TARC and macrophage inflammatory protein-1 beta as novel functional ligands for the CCR8 receptor. J Immunol. 1998; 28(2):582-588. (Biology). View Reference
  2. Combadiere C, Ahuja SK, Tiffany HL, Murphy PM. Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1(alpha), MIP-1(beta), and RANTES. J Leukoc Biol. 1996; 60(1):147-152. (Biology). View Reference
  3. Lipes MA, Napolitano M, Jeang KT, Chang NT, Leonard WJ. Identification, cloning, and characterization of an immune activation gene. Proc Natl Acad Sci U S A. 1988; 85(24):9704-9708. (Biology). View Reference
  4. Napolitano M, Seamon KB, Leonard WJ. Identification of cell surface receptors for the Act-2 cytokine. J Exp Med. 1990; 172(1):285-289. (Biology). 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
  6. Raport CJ, Gosling J, Schweickart VL, Gray PW, Charo IF. Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha. J Biol Chem. 1996; 271(29):17161-17166. (Biology). View Reference
  7. Samson M, Labbe O, Mollereau C, Vassart G, Parmentier M. Molecular cloning and functional expression of a new human CC-chemokine receptor gene. Biochemistry. 1996; 35(11):3362-3367. (Biology). View Reference
  8. Sherry B, Tekamp-Olson P, Gallegos C. one of those components, macrophage inflammatory protein 1 beta. J Exp Med. 1988; 168(6):2251-2259. (Biology). View Reference
  9. Ziegler SF, Tough TW, Franklin TL, Armitage RJ, Alderson MR. Induction of macrophage inflammatory protein-1 beta gene expression in human monocytes by lipopolysaccharide and IL-7. J Immunol. 1991; 147(7):2234-2239. (Biology). View Reference
View All (9) View Less
550078 Rev. 2

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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


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For Research Use Only. Not for use in diagnostic or therapeutic procedures.