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PE Rat Anti-Human IL-2
PE Rat Anti-Human IL-2

Expression of IL-2 by stimulated CD3+ and CD3-human PBMC. Human PBMC were stimulated for 18 hours with PMA (Sigma, Cat. No. P-8139) and ionomycin (Sigma, Cat. No. I-0634), in the presence of BD GolgiStop™ (2 μl final concentration; Cat. No. 554724). The PBMC were stained with PE-Cy™5-anti-CD3 (Cat. No. 555334), fixed, permeabilized, and then stained with 20 μl of the PE Rat Anti-Human IL-2 antibody (Cat. No. 560902/559334/554566; left panel).

To demonstrate specificity of staining, the binding of PE-MQ1-17H12 was blocked by the preincubation of the conjugated antibody with recombinant human IL-2 (0.25 mg, Cat. No. 554603; middle panel), and by preincubation of the fixed/permeabilized cells with an excess of Purified Rat Anti-Human IL-2 (10 mg, Cat. No. 554563; right panel) prior to staining with the PE-MQ1-17H12 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and unlabeled antibody blocking specificity controls (right panel).

Expression of IL-2 by stimulated CD3+ and CD3-human PBMC. Human PBMC were stimulated for 18 hours with PMA (Sigma, Cat. No. P-8139) and ionomycin (Sigma, Cat. No. I-0634), in the presence of BD GolgiStop™ (2 μl final concentration; Cat. No. 554724). The PBMC were stained with PE-Cy™5-anti-CD3 (Cat. No. 555334), fixed, permeabilized, and then stained with 20 μl of the PE Rat Anti-Human IL-2 antibody (Cat. No. 560902/559334/554566; left panel).

To demonstrate specificity of staining, the binding of PE-MQ1-17H12 was blocked by the preincubation of the conjugated antibody with recombinant human IL-2 (0.25 mg, Cat. No. 554603; middle panel), and by preincubation of the fixed/permeabilized cells with an excess of Purified Rat Anti-Human IL-2 (10 mg, Cat. No. 554563; right panel) prior to staining with the PE-MQ1-17H12 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and unlabeled antibody blocking specificity controls (right panel).

Product Details
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BD Pharmingen™
IL2; Interleukin-2; T-cell growth factor; TCGF
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Rat IgG2a, κ
Human IL-2 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_395483
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.

Recommended Assay Procedures

Immunofluorescent Staining and Flow Cytometric Analysis: The PE-conjugated MQ1-17H12 antibody (Cat. No. 554567/560902/559334) can be used for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-2-producing cells within mixed cell populations (see image). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.5 µg mAb/1X10^6 cells)  For specific methodology, please visit the protocols section on our web site, http://www.bdbiosciences.com/resources/index.jsp

A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated MQ1-17H12 antibody with a molar excess of ligand (e.g., recombinant human IL-2; Cat No. 554603) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabelled MQ1-17H12 antibody (Cat. No. 554563) prior to staining. A suitable rat IgG2a isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is PE-R35-95 (Cat. No. 554689); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/1X10^6 cells).

Neutralization/Blocking: The NA/LE™ format of the MQ1-17H12 antibody (Cat. No. 554652) is useful for neutralization of human IL-2 bioactivity. A suitable NA/LE™ rat IgG2a isotype control to match the NA/LE™ MQ1-17H12 antibody is the R35-95 antibody, Cat. No. 554687.

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. Cy is a trademark of GE Healthcare.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
554566 Rev. 4
Antibody Details
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MQ1-17H12

The MQ1-17H12 monoclonal antibody specifically binds to the multifunctional cytokine, human Interleukin-2 (IL-2). IL-2 is produced by activated T cells and has multiple functions that can affect the growth, proliferation, differentiation and survival of many different target cell types including T cells, B cells, NK cells, monocytes and macrophages. The immunogen used to generate the MQ1-17H12 hybridoma was purified recombinant human IL-2 protein. The MQ1-17H12 antibody reportedly neutralizes the biological activity of human IL-2.

554566 Rev. 4
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
554566 Rev.4
Citations & References
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Development References (2)

  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. (Biology). 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. (Biology). View Reference
554566 Rev. 4

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