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APC Rat Anti-Mouse IL-6
APC Rat Anti-Mouse IL-6

Flow cytometric analysis of intracellular IL-6 expression by activated mouse macrophages. Thioglycollate-elicited mouse peritoneal macrophages were primed with recombinant mouse IFN-γ (10 ng/ml, Cat. No. 554587) for 2 hr and stimulated overnight with lipopolysaccharide (LPS, Sigma, Cat. No. L-8272; 1 μg/ml) and BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (Cat. No. 555029). The adherent cells were washed with 1× phosphate buffered saline (PBS) and incubated with 1× trypsin-EDTA solution (37°C, 15 min). The cells were harvested, washed, incubated with Fc Block™ (Rat IgG2b,κ Anti-Mouse CD16/CD32) antibody (Cat. No. 553142), fixed and permeabilized using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained either with an APC Rat IgG1, κ Isotype Control (Cat No. 554686, Left Panel) or with the APC Rat Anti-Mouse IL-6 antibody (Cat No. 561367, Right Panel). MiCK-3 Mouse Cytokine Positive Control Cells (Cat No. 554654) are prepared in a similar manner. These cells can be used as a positive control for cytokine flow cytometry experiments designed to characterize the nature of mouse IL-6-producing cells. Two-color flow cytometric dot plots showing the correlated expression of IL-6 (or Ig Isotype control staining) versus cellular autofluorescence measured in the phycoerythrin channel (autofluorescence) were derived from events with the forward and side light-scatter characteristics of intact macrophages. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.

Flow cytometric analysis of intracellular IL-6 expression by activated mouse macrophages. Thioglycollate-elicited mouse peritoneal macrophages were primed with recombinant mouse IFN-γ (10 ng/ml, Cat. No. 554587) for 2 hr and stimulated overnight with lipopolysaccharide (LPS, Sigma, Cat. No. L-8272; 1 μg/ml) and BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (Cat. No. 555029). The adherent cells were washed with 1× phosphate buffered saline (PBS) and incubated with 1× trypsin-EDTA solution (37°C, 15 min). The cells were harvested, washed, incubated with Fc Block™ (Rat IgG2b,κ Anti-Mouse CD16/CD32) antibody (Cat. No. 553142), fixed and permeabilized using BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained either with an APC Rat IgG1, κ Isotype Control (Cat No. 554686, Left Panel) or with the APC Rat Anti-Mouse IL-6 antibody (Cat No. 561367, Right Panel). MiCK-3 Mouse Cytokine Positive Control Cells (Cat No. 554654) are prepared in a similar manner. These cells can be used as a positive control for cytokine flow cytometry experiments designed to characterize the nature of mouse IL-6-producing cells. Two-color flow cytometric dot plots showing the correlated expression of IL-6 (or Ig Isotype control staining) versus cellular autofluorescence measured in the phycoerythrin channel (autofluorescence) were derived from events with the forward and side light-scatter characteristics of intact macrophages. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.

Product Details
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BD Pharmingen™
Il6; Il-6; Interleukin-6; B-cell hybridoma growth factor
Mouse (QC Testing)
Rat IgG1
Mouse IL-6 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_10679354
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 to APC under optimum conditions, and unconjugated antibody and free APC were removed.

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. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser.
  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.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
561367 Rev. 1
Antibody Details
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MP5-20F3

The MP5-20F3 monoclonal antibody specifically binds to mouse interleukin-6 (IL-6). The immunogen used to generate the MP5-20F3 hybridoma was recombinant mouse IL-6.

561367 Rev. 1
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APC
Allophycocyanin (APC), is part of the BD family of phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 651 nm and an emission maximum (Em Max) at 660 nm. APC is designed to be excited by the Red (627-640 nm) laser and detected using an optical filter centered near 660 nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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APC
Red 627-640 nm
651 nm
660 nm
561367 Rev.1
Citations & References
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Development References (6)

  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: 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: Flow cytometry, IC/FCM Block). View Reference
  4. Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods. 1993; 166(2):201-214. (Biology: ELISA, Flow cytometry). View Reference
  5. Starnes HF Jr, Pearce MK, Tewari A, Yim JH, Zou JC, Abrams JS. Anti-IL-6 monoclonal antibodies protect against lethal Escherichia coli infection and lethal tumor necrosis factor-alpha challenge in mice. J Immunol. 1990; 145(12):4185-4191. (Biology: Flow cytometry, IC/FCM Block, Neutralization). View Reference
  6. Suda T, O'Garra A, MacNeil I, Fischer M, Bond MW, Zlotnik A. Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas. Cell Immunol. 1990; 129(1):228-240. (Biology: Neutralization). View Reference
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561367 Rev. 1

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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


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.