PE Rat Anti-Mouse IL-17A

BD Pharmingen™ PE Rat Anti-Mouse IL-17A

Name: PE Rat Anti-Mouse IL-17A
Brand: BD Pharmingen™
SKU: 559502

Clone TC11-18H10

(RUO)

Characterization of IL-17A-producing cells within a stimulated mouse EL4 thymoma cell population. EL4 cells were cultured overnight in the presence of GolgiPlug™ (1 µg/ml final concentration; Cat. No.555029) without (left panel) or with PMA (5 ng/ml final concentration; Sigma, Cat. No.P-8139) and Ionomycin (500 ng/ml final concentration; Sigma, Cat. No.I-0634) (middle and right panels). The cells were fixed, permeabilized and subsequently stained with 0.125 µg of PE conjugated anti-mouse IL-17A (PE-TC11-18H10, Cat. No.559502) using Pharmingen's I/C staining protocol (left and middle panels). To demonstrate the specificity of staining, the binding of PE-TC11-18H10 was blocked by the preincubation of the fixed/permeabilized, stimulated EL4 cells with unlabeled TC11-18H10 antibody (10 µg; Cat. No. 559501, right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the unstimulated stained (left panel) and unlabeled antibody blocking (right panel) controls.

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Product Details

Brand: BD Pharmingen™

Reactivity: Mouse (QC Testing)

Isotype: Rat LEW, also known as Lewis IgG1, κ

Immunogen: Recombinant Mouse IL-17A Protein

Application: Intracellular staining (flow cytometry) (Routinely Tested)

Concentration: 0.2 mg/ml

RRID: AB_397256

Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide.

Regulatory Status: 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

Recommended Assay Procedure:

Immunofluorescent Staining and Flow Cytometric Analysis: The TC11-18H10 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-17A producing cells within mixed cell populations. The PE-conjugated TC11-18H10 antibody (Cat. No. 559502) is especially suitable for these experiments (see image). For optimal immunofluorescent staining and flow cytometric analysis, this anti-cytokine antibody should be titrated (≤ 0.25 µg mAb/million cells). For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining in the Immune Function Handbook..

A useful control for demonstrating the specificity of staining is to pre-block paraformaldehyde-fixed/saponin-permeabilized target cells with unlabeled TC11-18H10 antibody (Cat. No. 559501) prior to staining. The intracellular staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse is PE-R3-34 (Cat. No. 554685); use at comparable concentrations to antibody of interest (e.g., ≤ 0.25 µg mAb/1 million cells).

OTHER APPLICATIONS

1. ELISA Capture: The purified TC11-18H10 antibody (Cat. No. 555068) is useful as a capture antibody for a sandwich ELISA for measuring mouse IL-17A protein levels. Purified TC11-18H10 antibody can be paired with the biotinylated TC11-8H4 (Cat. No.555067) antibody as the detecting antibody, with recombinant mouse IL-17A protein as the standard. The purified TC11-18H10 antibody should be titrated from 0.5 µg/ml to 2.0 µg/ml to determine its optimal concentration for ELISA capture. To obtain linear standard curves, doubling dilutions of mouse IL-17A ranging from ~2,000 to 15 pg/ml are recommended for inclusion in each ELISA plate. For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on ELISA in the Immune Function Handbook.

2. Western blotting: The TC11-1810 antibody has been found useful for Western Blotting. Please note that this application is not routinely tested at BD Biosciences Pharmingen.

3. Neutralization: TCII-18H10 has been shown to be a neutralizing antibody. Please note that this application is not routinely tested at BD Biosciences.

Product Notices

Since applications vary, each investigator should titrate the reagent to obtain optimal results.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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.

Companion Products

PE Rat IgG1, κ Isotype Control RUO

Size 0.1 mg Cat No. 554685

Protein Transport Inhibitor (Containing Brefeldin A) RUO

Size 1 mL Cat No. 555029

559502 Rev. 2

Antibody Details

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.

559502 Rev. 2

Format Details

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.

Format: PE

Excitation Source: Yellow-Green 488 nm, 532 nm, 561 nm

Excitation Max: 496 nm, 566 nm

Emission Max: 576 nm

559502 Rev.2

Citations & References

Development References (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
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
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

559502 Rev. 2

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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.