PE Mouse Anti-Rat IL-10

BD Pharmingen™ PE Mouse Anti-Rat IL-10

Name: PE Mouse Anti-Rat IL-10
Brand: BD Pharmingen™
SKU: 555088

Clone A5-4

(RUO)

Expression of IL-10 by stimulated CD4+ rat splenocytes. Purified splenic CD4+ cells from 6 month old Lewis rats were stimulated with plate-bound anti-CD3 (25 µg/ml final concentration; G4.18, Cat. No. 554829) and soluble anti-rat CD28 (2 µg/ml final concentration; clone JJ319, Cat. No. 554993) for 2 days in culture together with rat IL- 2 (10 ng/ml final concentration; Cat. No. 555106) and rat IL-4 (10 ng/ml final concentration; Cat. No 555107), followed by a 3 day incubation with only rat IL-2 and rat IL-4. This was followed by a 6 hour stimulation with PMA (1 ng/ml final concentration; Sigma, Cat. #P-8139) and Ionomycin (250 ng/ml final concentration; Sigma, Cat. #I-0634) in the presence of BD GolgiStop ™ (2 µM final concentration Cat. No. 554724). The cells were then fixed, permeabilized, and subsequently stained with 0.06 µg of PE Mouse anti rat IL-10 antibody (PE-A5-4, Cat. No. 555088; Left panel) by using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of the PE-A5-4 antibody was blocked by preincubation of the PE- conjugated antibody with recombinant rat IL-10 (0.25 µg, Cat. No. 555113; Second panel), and by preincubation of the fixed/permeabilized cells with the unlabeled A5-4 antibody (10 µg; Right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (Center panel) and unlabeled antibody blocking (Right panel) specificity controls.

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Product Details

Brand: BD Pharmingen™

Alternative Name: Il10; Interleukin-10; CSIF; Cytokine synthesis inhibitory factor

Reactivity: Rat (QC Testing)

Isotype: Mouse IgG2b, κ

Immunogen: Recombinant Rat IL-10

Application: Intracellular block/flow cytometry (Routinely Tested)

Concentration: 0.2 mg/ml

RRID: AB_395691

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

Regulatory Status: 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

Immunoflourecent Staining and Flow Cytometric Analysis: The PE-conjugated A5-4 antibody (Cat. No. 555088) can be used for multicolor immunofluorescent staining and flow cytometric analyses to identify and enumerate IL-10-producing cells within mixed cell populations (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 our web site, w ww.bdbiosciences.com , and go to the protocols section or the chapter on intracellular staining in the Immune Function Handbook. An appropriate isotype control is Cat. No. 555058.

A useful control for demonstrating specificity of staining is either of the following: 1) preblock the conjugated A5-4 antibody with a ligand (e.g., recombinant rat IL-10; Cat. No. 555113) prior to staining, or 2) pre-block the fixed/permeabilized cells with unconjugated A5-4 antibody prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.

Product Notices

Since applications vary, each investigator should titrate the reagent to obtain optimal results.
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.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
An isotype control should be used at the same concentration as the antibody of interest.

555088 Rev. 3

Antibody Details

A5-4

The A5-4 monoclonal antibody specifically binds to rat interleukin-10 (IL-10). The immunogen used to generate the A5-4 hybridoma was recombinant rat IL-10 protein.

555088 Rev. 3

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

555088 Rev.3

Citations & References

Development References (2)

Feng L, Tang WW, Chang JC, Wilson CB. Molecular cloning of rat cytokine synthesis inhibitory factor (IL-10) cDNA and expression in spleen and macrophages. Biochem Biophys Res Commun. 1993; 192(2):452-458. (Immunogen). 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

555088 Rev. 3

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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.