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BD Pharmingen™ PE Rat Anti-Human IL-3
Clone BVD3-1F9 (RUO)
Expression of IL-3 by stimulated human CD4 cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 mouse antibody (UCHT1, Cat. No. 555329), soluble anti-human CD28 mouse antibody (CD28.2, Cat. No. 555725), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL- 4 (20 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL- 4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hours with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (1 µg/ml final concentration; Sigma, Cat. #C-9275) in the presence of Golgi-Stop™ (2 µM final concentration; Cat. #554724). The cells were harvested, stained with Pe-Cy5 anti- CD4 (RPA-T4, Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-rat anti-human IL-3 antibody (PE-BVD3-1F9, Cat. No. 554676) (see Left panel). To demonstrate specificity of staining, the binding by PE- BVD3-1F9 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with 0.5µg recombinant human IL-3 (see Center panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled BVD3-1F9 antibody (5 µg; Cat. No. 554673; see Right panel) prior to staining with the PE-BVD3- 1F9 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Expression of IL-3 by stimulated human CD4 cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 mouse antibody (UCHT1, Cat. No. 555329), soluble anti-human CD28 mouse antibody (CD28.2, Cat. No. 555725), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL- 4 (20 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL- 4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hours with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (1 µg/ml final concentration; Sigma, Cat. #C-9275) in the presence of Golgi-Stop™ (2 µM final concentration; Cat. #554724). The cells were harvested, stained with Pe-Cy5 anti- CD4 (RPA-T4, Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-rat anti-human IL-3 antibody (PE-BVD3-1F9, Cat. No. 554676) (see Left panel). To demonstrate specificity of staining, the binding by PE- BVD3-1F9 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with 0.5µg recombinant human IL-3 (see Center panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled BVD3-1F9 antibody (5 µg; Cat. No. 554673; see Right panel) prior to staining with the PE-BVD3- 1F9 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Expression of IL-3 by stimulated human CD4 cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 mouse antibody (UCHT1, Cat. No. 555329), soluble anti-human CD28 mouse antibody (CD28.2, Cat. No. 555725), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL- 4 (20 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL- 4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hours with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (1 µg/ml final concentration; Sigma, Cat. #C-9275) in the presence of Golgi-Stop™ (2 µM final concentration; Cat. #554724). The cells were harvested, stained with Pe-Cy5 anti- CD4 (RPA-T4, Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-rat anti-human IL-3 antibody (PE-BVD3-1F9, Cat. No. 554676) (see Left panel). To demonstrate specificity of staining, the binding by PE- BVD3-1F9 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with 0.5µg recombinant human IL-3 (see Center panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled BVD3-1F9 antibody (5 µg; Cat. No. 554673; see Right panel) prior to staining with the PE-BVD3- 1F9 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Expression of IL-3 by stimulated human CD4 cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 mouse antibody (UCHT1, Cat. No. 555329), soluble anti-human CD28 mouse antibody (CD28.2, Cat. No. 555725), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL- 4 (20 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL- 4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hours with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (1 µg/ml final concentration; Sigma, Cat. #C-9275) in the presence of Golgi-Stop™ (2 µM final concentration; Cat. #554724). The cells were harvested, stained with Pe-Cy5 anti- CD4 (RPA-T4, Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-rat anti-human IL-3 antibody (PE-BVD3-1F9, Cat. No. 554676) (see Left panel). To demonstrate specificity of staining, the binding by PE- BVD3-1F9 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with 0.5µg recombinant human IL-3 (see Center panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled BVD3-1F9 antibody (5 µg; Cat. No. 554673; see Right panel) prior to staining with the PE-BVD3- 1F9 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Product Details
BD Pharmingen™
Human (QC Testing)
Rat IgG1
Recombinant Human IL-3
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_395504
Aqueous buffered solution containing ≤0.09% sodium azide.
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
Immunofluorescent Staining and Flow Cytometric Analysis: The BVD3-1F9 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-3 producing cells within mixed cell populations. The PE-conjugated BVD3-1F9 antibody (Cat. No. 554676) is especially suitable for these studies. For optimal immunofluorescent staining and flow cytometric analysis, this anti-cytokine antibody should be titrated ( ≤ 0.5 µg mAb/million cells). For specific methodology, please visit the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.
A suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells is R3-34 (Cat. No. 554685); use at comparable concentrations to antibody of interest (e.g., ≤ 0.5 µg mAb/1 million cells). A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the conjugated BVD3-1F9 antibody with ligand (e.g., recombinant human IL-3; Cat No. 554604) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabeled BVD3-1F9 antibody prior to staining. The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.
ELISA: The biotinylated BVD3-1F9 antibody (Cat. No. 554674) is useful as a detection antibody for a sandwich ELISA for measuring human IL-3 protein levels. Biotinylated BVD3-1F9 antibody can be paired with the purified BVD8-3G11 antibody (Cat. No. 554672) as the capture antibody, with recombinant human IL-3 protein (Cat. No. 554604) as the standard.
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.
554676 Rev. 1
Antibody Details
BVD3-1F9
The BVD3-1F9 antibody reacts with human interleukin-3 (IL-3). The immunogen used to generate the BVD3-1F9 hybridoma was recombinant human IL-3. This is a weakly neutralizing antibody.
554676 Rev. 1
Format Details
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.
PE
496 nm, 566 nm
576 nm
554676 Rev.1
Citations & References
View product citations for antibody "554676" on CiteAb
Development References (5)
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Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
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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. (Clone-specific: ELISA). View Reference
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Abrams JS, Silver JE, Van Dyke RE, Gleich GI. Eosinophil-active cytokines in human disease: development and use of monoclonal antibodies to IL-3, IL-5 and GMCSF. In: Gleich GJ and Kay AB, ed. Eosinophils in Allergy and Inflammation. New York: Dekker; 1994:133-157.
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Kaushansky K, Shoemaker SG, Broudy VC. Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3. J Clin Invest. 1992; 90(5):1879-1888. (Clone-specific). View Reference
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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
554676 Rev. 1
Please refer to Support Documents for Quality Certificates
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.
Please refer to Support Documents for Quality Certificates
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.