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560821Image1.png

Flow cytometric analysis of IL-17A-producing cells within a stimulated mouse EL4 thymoma cell population. EL4 cells were stimulated (left and middle panels) or unstimulated (right panel) with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and Ionomycin (1000 ng/ml final concentration; Sigma, Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724) for 5 hours. The cells were fixed, permeabilized and subsequently stained with APC-Cy™7 Rat Anti-Mouse IL-17A (Cat. No. 560821; middle and right panels) or APC-Cy™7  Rat IgG1, κ Isotype Control (Cat. No. 560534; left panel) using the "BD Cytofix/Cytoperm Method for Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis". Bivariate flow cytometric dot plots showing correlated expression patterns of IL-17A (APC-Cy™7) versus cellular autofluorescence (FL1) were derived from gated events with the forward and side light-scatter characteristics of viable cells. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control (FL1) and the APC-Cy™7  Rat IgG1, κ Isotype Control staining. Flow cytometry was performed on a BD™ LSR II Flow Cytometer System.

560821Image1.png

Flow cytometric analysis of IL-17A-producing cells within a stimulated mouse EL4 thymoma cell population. EL4 cells were stimulated (left and middle panels) or unstimulated (right panel) with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and Ionomycin (1000 ng/ml final concentration; Sigma, Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724) for 5 hours. The cells were fixed, permeabilized and subsequently stained with APC-Cy™7 Rat Anti-Mouse IL-17A (Cat. No. 560821; middle and right panels) or APC-Cy™7  Rat IgG1, κ Isotype Control (Cat. No. 560534; left panel) using the "BD Cytofix/Cytoperm Method for Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis". Bivariate flow cytometric dot plots showing correlated expression patterns of IL-17A (APC-Cy™7) versus cellular autofluorescence (FL1) were derived from gated events with the forward and side light-scatter characteristics of viable cells. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control (FL1) and the APC-Cy™7  Rat IgG1, κ Isotype Control staining. Flow cytometry was performed on a BD™ LSR II Flow Cytometer System.

560821Image1.png 560821Image1.png mIL-17a_APC_Cy7_untreated_EL4.png Red Cap_UV.png

Flow cytometric analysis of IL-17A-producing cells within a stimulated mouse EL4 thymoma cell population. EL4 cells were stimulated (left and middle panels) or unstimulated (right panel) with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and Ionomycin (1000 ng/ml final concentration; Sigma, Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724) for 5 hours. The cells were fixed, permeabilized and subsequently stained with APC-Cy™7 Rat Anti-Mouse IL-17A (Cat. No. 560821; middle and right panels) or APC-Cy™7  Rat IgG1, κ Isotype Control (Cat. No. 560534; left panel) using the "BD Cytofix/Cytoperm Method for Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis". Bivariate flow cytometric dot plots showing correlated expression patterns of IL-17A (APC-Cy™7) versus cellular autofluorescence (FL1) were derived from gated events with the forward and side light-scatter characteristics of viable cells. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control (FL1) and the APC-Cy™7  Rat IgG1, κ Isotype Control staining. Flow cytometry was performed on a BD™ LSR II Flow Cytometer System.

Flow cytometric analysis of IL-17A-producing cells within a stimulated mouse EL4 thymoma cell population. EL4 cells were stimulated (left and middle panels) or unstimulated (right panel) with PMA (50 ng/ml final concentration; Sigma, Cat. No. P-8139) and Ionomycin (1000 ng/ml final concentration; Sigma, Cat. No. I-0634) in the presence of BD GolgiStop™ Protein Transport Inhibitor (Cat. No. 554724) for 5 hours. The cells were fixed, permeabilized and subsequently stained with APC-Cy™7 Rat Anti-Mouse IL-17A (Cat. No. 560821; middle and right panels) or APC-Cy™7  Rat IgG1, κ Isotype Control (Cat. No. 560534; left panel) using the "BD Cytofix/Cytoperm Method for Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis". Bivariate flow cytometric dot plots showing correlated expression patterns of IL-17A (APC-Cy™7) versus cellular autofluorescence (FL1) were derived from gated events with the forward and side light-scatter characteristics of viable cells. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control (FL1) and the APC-Cy™7  Rat IgG1, κ Isotype Control staining. Flow cytometry was performed on a BD™ LSR II Flow Cytometer System.

Product Details
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BD Pharmingen™
IL-17; Il17; IL-17A; Il17a; CTLA-8; Ctla8; Interleukin-17A; interleukin 17A
Mouse (QC Testing)
Rat LEW, also known as Lewis IgG1, κ
Recombinant Mouse IL-17A Protein
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_2034016
Aqueous buffered solution containing protein stabilizer 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 APC-Cy7 under optimum conditions, and unconjugated antibody and free APC-Cy7 were removed.
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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. Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
  5. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
  6. APC-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher.
  7. APC-Cy7 is a tandem fluorochrome composed of Allophycocyanin (APC), which is excited by laser lines between 595 and 647 nm and serves as an energy donor, coupled to the cyanine dye Cy7™, which acts as an energy acceptor and fluoresces at 780 nm. BD Biosciences Pharmingen has maximized the fluorochrome energy transfer in APC-Cy7, thus maximizing its fluorescence emission intensity, minimizing residual emission from APC, and minimizing required electronic compensation in multilaser-laser flow cytometry systems. Note: Although every effort is made to minimize the lot-to-lot variation in residual emission from APC, it is strongly recommended that every lot be tested for differences in the amount of compensation required and that individual compensation controls are run for each APC-Cy7 conjugate.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Cy is a trademark of GE Healthcare.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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560821 Rev. 2
Antibody Details
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TC11-18H10

The TC11-18H10 monoclonal antibody specifically binds to recombinant and natural mouse IL-17A proteins. IL-17A, also known as CTLA-8, is a T cell-derived cytokine that promotes inflammatory responses.  Mouse IL-17A is a proinflammatory cytokine that can induce the release of IL-6 by mouse stromal cells. It has been shown to support the growth of hemopoietic progenitors in vitro; it can also stimulate granulopoiesis in vivo. The TC11-18H10 antibody has been reported to neutralize IL-17A activity.  Recent studies have shown that IL-17A is produced by a unique subset of Th17 cells that develop along a pathway distinct from the Th1- and Th2- cell differentiation pathways. The mouse IL-17A cDNA was isolated from a cDNA library generated from TCRαβ+CD4-CD8- thymocytes.

560821 Rev. 2
Format Details
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APC-Cy7
APC-Cy7 dye is a part of the BD APC red family of dyes. This tandem fluorochrome is comprised of a Allophycocyanin (APC) donor that has excitation maxima (Ex Max) of 651 nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 779 nm. APC-Cy7 can be excited by the Red (627-640 nm) laser and detected using an optical filter centered near 780 nm (e.g., a 760/60 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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APC-Cy7
651 nm
779 nm
560821 Rev.2
Citations & References
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View product citations for antibody "560821" on CiteAb

Development References (7)

  1. Coquet JM, Chakravarti S, Kyparissoudis K, et al. Diverse cytokine production by NKT cell subsets and identification of an IL-17-producing CD4-NK1.1- NKT cell population. Proc Natl Acad Sci U S A. 2008; 105(32):11287-11292. (Clone-specific). View Reference
  2. Dong C. Th17 cells: Current understanding of their generation and regulation. Eur J Immunol. 2009; 39(3):640-644. (Biology). View Reference
  3. Kennedy J, Rossi DL, Zurawski SM, et al. Mouse IL-17: a cytokine preferentially expressed by alpha beta TCR + CD4-CD8-T cells. J Interferon Cytokine Res. 1996; 16(8):611-617. (Biology). View Reference
  4. Lampropoulou V, Hoehlig K, Roch T, et al. TLR-activated B cells suppress T cell-mediated autoimmunity. J Immunol. 2008; 180(7):4763-4773. (Clone-specific). 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). View Reference
  6. Schwarzenberger P, La Russa V, Miller A, et al. IL-17 stimulates granulopoiesis in mice: use of an alternate, novel gene therapy-derived method for in vivo evaluation of cytokines. J Immunol. 1998; 161(11):6383-6389. (Biology). View Reference
  7. Yen D, Cheung J, Scheerens H et al. IL-23 is essential for T cell–mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest. 2006; 116(5):1310-1316. (Biology). View Reference
View All (7) View Less
560821 Rev. 2

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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.