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PE Rat Anti-Mouse IL-10
PE Rat Anti-Mouse IL-10

Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 (clone 145-2C11, Cat. No. 553058 at 25 µg/ml) and soluble anti-mouse CD28 (clone 37.51, Cat. No. 553294 at 2 µg/ml) for 2 days in culture together with recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and recombinant mouse IL-4 (1 ng/ml, Cat. No. 550067), followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 (25 µg/ml) and anti-mouse CD28 (2 µg/ml) in the presence of GolgiStop™ (Cat. No. 554724). The cells were then stained with 0.06 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553046) and 0.12 µg of PE-conjugated rat anti-mouse IL-10 antibody (PE-JES5-16E3, Cat. No. 554467) by using BD Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 (0.12 µg, Cat. No.550070; middle panel), and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb (3.6 µg, Cat. No. 554464; right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and unlabelled antibody blocking (right panel) specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 (Cat. No. 556925); use at comparable concentrations to antibody of interest.

Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 (clone 145-2C11, Cat. No. 553058 at 25 µg/ml) and soluble anti-mouse CD28 (clone 37.51, Cat. No. 553294 at 2 µg/ml) for 2 days in culture together with recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and recombinant mouse IL-4 (1 ng/ml, Cat. No. 550067), followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 (25 µg/ml) and anti-mouse CD28 (2 µg/ml) in the presence of GolgiStop™ (Cat. No. 554724). The cells were then stained with 0.06 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553046) and 0.12 µg of PE-conjugated rat anti-mouse IL-10 antibody (PE-JES5-16E3, Cat. No. 554467) by using BD Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 (0.12 µg, Cat. No.550070; middle panel), and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb (3.6 µg, Cat. No. 554464; right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and unlabelled antibody blocking (right panel) specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 (Cat. No. 556925); use at comparable concentrations to antibody of interest.

製品詳細
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BD Pharmingen™
Mouse (QC Testing)
Rat IgG2b
Recombinant mouse IL-10
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_395412
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


調製と保管

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.

推奨アッセイ手順

The PE-conjugated JES5-16E3 antibody can be used for multicolor 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, visit our web site, www.bdbiosciences.com, and go to the protocols section or the Techniques for Immune Function Analysis Application Handbook, Chapter 4: Immunofluorescent Staining of Intracellular Molecules for Flow Cytometric Analysis.

製品通知

  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.

コンパニオン製品

Protein Transport Inhibitor (Containing Monensin) RUO
サイズ 0.7 mL カタログ番号 554724
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PE Rat IgG2b, κ Isotype Control RUO
サイズ 0.1 mg カタログ番号 556925
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Purified Hamster Anti-Mouse CD3e RUO
サイズ 0.5 mg カタログ番号 553058
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Purified NA/LE Hamster Anti-Mouse CD28 RUO
サイズ 0.5 mg カタログ番号 553294
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Recombinant Mouse IL-2 RUO
サイズ 20 µg カタログ番号 550069
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Recombinant Mouse IL-4 RUO
サイズ 10 µg カタログ番号 550067
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554467 Rev. 2
抗体の詳細
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JES5-16E3

The JES5-16E3 monoclonal antibody specifically binds to the mouse cytokine, Interleukin-10 (IL-10). IL-10 is also known as Cytokine Synthesis Inhibitory Factor (CSIF). It is produced by various activated cell types including CD4+ T cells, CD8+ T cells, T regulatory cells, NK T cells, B1 B cells, NK cells, macrophages, dendritic cells, mast cells, granulocytes and keratinocytes. IL-10 plays a pivotal role in regulating immune responses and protecting the host from damage caused by inflammatory and autoimmune responses. IL-10 has numerous biological activities including the inhibition of cytokine synthesis by activated T cells, NK cells, monocytes, and macrophages. In the presence of accessory cells, IL-10 inhibits mitogen- or anti-CD3 induced proliferation of T lymphocytes.  IL-10 has also been shown to costimulate the development of thymocytes, B cell differentiation and the generation of cytotoxic T cells. The immunogen used to generate the JES5-16E3 hybridoma was recombinant mouse IL-10. JES5-16E3 is a neutralizing antibody.

554467 Rev. 2
フォーマットの詳細
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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
554467 Rev.2
引用&参考文献
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開発者向け参考資料 (3)

  1. 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.
  2. Litton MJ, Sander B, Murphy E, O'Garra A, Abrams JS. Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B. J Immunol Methods. 1994; 175(1):47-58. (Clone-specific: Neutralization). 参考文献を見る
  3. 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. (Clone-specific: Immunocytochemistry (cytospins), Neutralization). 参考文献を見る
554467 Rev. 2

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