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PE Mouse Anti-Human MCP-1
PE Mouse Anti-Human MCP-1

Expression of MCP-1 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 24 hours with LPS (10 ng/ml final concentration) in the presence of BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with PE Mouse Anti-Human CD14 (Cat. No. 555397), fixed, permeabilized, and subsequently stained with 20 µl of PE Mouse Anti-Human MCP-1 (Cat. No. 559324/554666/557066) by following the Usage section below and BD Biosciences 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, binding by PE Mouse Anti-Human MCP-1 was blocked by preincubation of the fixed/permeabilized cells with excess unlabeled Purified Mouse Anti-Human MCP-1 (5 µg; Cat. No. 554662; middle panel) prior to staining with PE Mouse Anti-Human MCP-1. The level of non-specific staining was assessed using the PE Mouse IgG1, κ Isotype Control (0.25 µg; Cat. No. 554680; right panel). 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 MCP-1 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 24 hours with LPS (10 ng/ml final concentration) in the presence of BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with PE Mouse Anti-Human CD14 (Cat. No. 555397), fixed, permeabilized, and subsequently stained with 20 µl of PE Mouse Anti-Human MCP-1 (Cat. No. 559324/554666/557066) by following the Usage section below and BD Biosciences 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, binding by PE Mouse Anti-Human MCP-1 was blocked by preincubation of the fixed/permeabilized cells with excess unlabeled Purified Mouse Anti-Human MCP-1 (5 µg; Cat. No. 554662; middle panel) prior to staining with PE Mouse Anti-Human MCP-1. The level of non-specific staining was assessed using the PE Mouse IgG1, κ Isotype Control (0.25 µg; Cat. No. 554680; right panel). 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™
CCL2; C-C motif chemokine 2; Chemokine (C-C motif) ligand 2; MCAF; SCYA2
Human (QC Testing), Rhesus, Cynomolgus (Tested in Development)
Mouse IgG1, κ
Recombinant Human MCP-1
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_397221
Aqueous buffered solution containing BSA and ≤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.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis:A useful control for demonstrating specificity of staining is to pre-block the paraformaldehyde-fixed/saponin-permeabilized cells with Purified Mouse Anti-Human MCP-1 (Cat. No. 551226) prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is also available in a 100 Test Size formulation PE Mouse IgG1, κ Isotype Control (Cat. No. 559320). For specific methodology, please visit the protocols section of "Intracellular Flow" on our web site, http://www.bdbiosciences.com/us/s/resources.

Important Note: This pre-titered antibody solution does not contain a cell permeabilization agent. It is necessary to include a cell permeabilization agent when using the pre-titered antibody solution to stain fixed and permeabilized cells. BD Perm/Wash™ Buffer (Cat. No. 554723) contains the permeabilization agent saponin and is useful for this purpose as described in the Usage section below.

Usage

1. Resuspend 1 × 10^6 fixed (BD Cytofix/Cytoperm™, Cat. No. 554722) and permeabilized cells in 20 µl of the pre-titered antibody solution and 30 µl of 1× BD Perm/Wash™ Buffer (Cat. No. 554723).

2. Incubate the cell suspension for 15 minutes (at RT or 4°C).

3. Wash twice in 100 µl of 1× BD Perm/Wash™ Buffer (Cat. No. 554723).

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. 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.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
559324 Rev. 3
Antibody Details
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5D3-F7

The 5D3-F7 monoclonal antibody specifically binds to human monocyte chemoattractant protein-1 (MCP-1), also known as CCL2 (C-C motif chemokine 2), Monocyte chemotactic and activating factor (MCAF), and Small-inducible cytokine A2 (SCYA2). MCP-1 is a 10-14 kDa glycoprotein member of the beta or CC family of chemokines. It expressed by monocytes, fibroblasts, endothelial cells and other cell types in response to IL-1, IL-6, TNF, and a variety of other stimuli. MCP-1 binds to and exerts its biological activity through G-protein coupled chemokine receptors including CCR2/CD192 and CCR4/CD194. It serves as a chemoattractant and activating factor for monocytes and other cell types including T cells, NK cells, and basophils.

MCP-1 is a member of the CC chemokine family and it is produced by monocytes, T lymphocytes, fibroblasts, endothelial cells, smooth muscle cells, keratinocytes and some tumors. Its production can be induced by LPS or IFN-γ. Clone 5D3-F7 also cross reacts with an intracellular component of LPS-stimulated (24 hours) peripheral blood monocytes of rhesus and cynomolgus macaque monkeys. The staining pattern observed on non-human primate monocytes is not as strong as that seen on normal human peripheral blood monocytes.

559324 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.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
559324 Rev.3
Citations & References
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Development References (2)

  1. Peri G, Milanese C, Matteucci C, et al. A new monoclonal antibody (5D3-F7) which recognizes human monocyte-chemotactic protein-1 but not related chemokines. Development of a sandwich ELISA and in situ detection of producing cells. J Immunol Methods. 1994; 174(1-2):249-257. (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. (Clone-specific: Flow cytometry). View Reference
559324 Rev. 3

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