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PE Rat Anti-Human IL-6
PE Rat Anti-Human IL-6

Expression of IL-6 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (100 ng/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with FITC-mouse anti-human CD14 monoclonal antibody (FITC-M5E2, Cat. No. 555397), fixed, permeabilized, and subsequently stained with 0.25 µg of PE-rat anti-human IL-6 antibody (PE-MQ2-6A3, Cat. No. 554697), following the BD Pharmingen staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scatter. To demonstrate specificity of staining, the binding by PE-MQ2-6A3 was blocked by each of the following: 1) preincubation of the conjugated antibody with recombinant human IL-6 (0.5 µg, Cat. No. 550071; middle panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled MQ2-6A3 antibody (2.5 µg; Cat. No. 559068, right panel) prior to staining with the PE-MQ2-6A3. 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.

Expression of IL-6 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (100 ng/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with FITC-mouse anti-human CD14 monoclonal antibody (FITC-M5E2, Cat. No. 555397), fixed, permeabilized, and subsequently stained with 0.25 µg of PE-rat anti-human IL-6 antibody (PE-MQ2-6A3, Cat. No. 554697), following the BD Pharmingen staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scatter. To demonstrate specificity of staining, the binding by PE-MQ2-6A3 was blocked by each of the following: 1) preincubation of the conjugated antibody with recombinant human IL-6 (0.5 µg, Cat. No. 550071; middle panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled MQ2-6A3 antibody (2.5 µg; Cat. No. 559068, right panel) prior to staining with the PE-MQ2-6A3. 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™
Human (QC Testing)
Rat IgG2a, κ
Recombinant human IL-6
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_395515
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

Immunofluorescent Staining and Flow Cytometric Analysis: The MQ2-6A3 antibody is useful for immunoflourescent staining and flow cytometric analysis to identify and enumerate human IL-6 producing cells within mixed cell populations. The PE- and FITC-conjugated MQ2-6A3 antibodies (Cat. No. 554696; 554697) are especially suitable for these experiments. (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 the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.

A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated MQ2-6A3 antibody with a ligand (e.g., recombinant human IL-6; Cat. No. 550071) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabelled MQ2-6A3 antibody prior to staining. The staining technique and blocking controls are described in detail by C. Prussin and D. Metcalfe. 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/1 million cells).

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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. 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.
554697 Rev. 1
Antibody Details
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MQ2-6A3

The MQ2-6A3 antibody reacts with human interleukin-6 (IL-6). The immunogen used to generate the MQ2-6A3 hybridoma was recombinant human IL-6. This is a neutralizing antibody.

554697 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
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Citations & References
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Development References (4)

  1. Andersson J, Abrams J, Bjork L, et al. Concomitant in vivo production of 19 different cytokines in human tonsils. Immunology. 1994; 83(1):16-24. (Clone-specific). View Reference
  2. Andersson U, Andersson J. Immunolabeling of cytokine-producing cells in tissues and in suspension. In: Fradelizie D, Emelie D, ed. Cytokine Producing Cells. Paris: Inserm; 1994:32-49.
  3. Litton M, Andersson J, Bjork L, Fehniger T, Ulfgren AK, Andersson U. Cytoplasmic cytokine staining in individual cells. In: Debets and Savelkoul, ed. Human Cytokine Protocols. Humana Press; 1996.
  4. 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
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554697 Rev. 1

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