Two-color flow cytometric analysis of IL-10 expression in stimulated Human lymphocytes. Human peripheral blood mononuclear cells were stimulated with immobilized Purified NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 µg/ml for plate coating) and soluble Purified NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 2 µg/ml) antibodies and Recombinant Human IL-2 (Cat. No. 554603; 10 ng/ml) and Human IL-4 (Cat. No. 554605; 25 ng/ml) proteins for 2 days. The stimulated cells were washed and cultured in medium containing IL-2 and IL-4 for another 3 days. Finally, the cells were harvested and cultured (5 hr) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) either without (Unstimulated; Left Plot) or with (Restimulated; Right Plot) Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed, permeabilized and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with BD Horizon™ RY586 Mouse Anti-Human IL-10 and Viral IL-10 antibody (Cat. No. 568520/568521; Right Plot) using BD Biosciences Intracellular Cytokine Staining protocol. Pseudocolor density plots showing the correlated expression of IL-10 versus side light-scatter signals (SSC-A) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 Cell Analyzer System and FlowJo™ software.
Two-color flow cytometric analysis of IL-10 expression in stimulated Human lymphocytes. Human peripheral blood mononuclear cells were stimulated with immobilized Purified NA/LE Mouse Anti-Human CD3 (Cat. No. 555329; 10 µg/ml for plate coating) and soluble Purified NA/LE Mouse Anti-Human CD28 (Cat. No. 555725; 2 µg/ml) antibodies and Recombinant Human IL-2 (Cat. No. 554603; 10 ng/ml) and Human IL-4 (Cat. No. 554605; 25 ng/ml) proteins for 2 days. The stimulated cells were washed and cultured in medium containing IL-2 and IL-4 for another 3 days. Finally, the cells were harvested and cultured (5 hr) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724) either without (Unstimulated; Left Plot) or with (Restimulated; Right Plot) Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were then washed, permeabilized and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with BD Horizon™ RY586 Mouse Anti-Human IL-10 and Viral IL-10 antibody (Cat. No. 568520/568521; Right Plot) using BD Biosciences Intracellular Cytokine Staining protocol. Pseudocolor density plots showing the correlated expression of IL-10 versus side light-scatter signals (SSC-A) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 Cell Analyzer System and FlowJo™ software.
Product Details
BD Horizon™
CSIF; Cytokine synthesis inhibitory factor; TGIF
Human (QC Testing), Viral (Tested in Development)
Rat IgG1
Recombinant Human IL-10
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
Aqueous buffered solution containing ≤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 to the dye under optimum conditions and unreacted dye was removed.
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- CF™ is a trademark of Biotium, Inc.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
Companion Products
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Protein Transport Inhibitor (Containing Monensin) RUO
Size
0.7 mL
Cat No.
554724
Protein Transport Inhibitor (Containing Brefeldin A) RUO
Size
1 mL
Cat No.
555029
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Antibody Details
JES3-9D7
The JES3-9D7 monoclonal antibody specifically reacts with human IL-10 (Interleukin-10) and viral IL-10. The immunogen used to generate the JES3-9D7 hybridoma was recombinant human IL-10 expressed by COS cells. IL-10 is also known as CSIF (Cytokine synthesis inhibitory factor) and (TGIF) T-cell growth inhibitory factor. IL-10 is expressed by various cell types including activated monocytes, macrophages, dendritic cells, mast cells, granulocytes, and lymphocytes. IL-10 is a pleiotropic cytokine that can downregulate proinflammatory immune responses, such as Th1-like responses, while promoting other responses including B cell proliferation and antibody production.
Format Details
RY586
The BD Horizon RealYellow™ 586 (RY586) Dye is part of the BD family of yellow-green dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 565-nm and an emission maximum (Em Max) at 586-nm. Driven by BD innovation, RY586 can be used on both spectral and conventional cytometers and is designed to be excited by the Yellow-Green laser (561-nm) with minimal excitation by the 488-nm Blue laser. For conventional instruments equipped with a Yellow-Green laser (561-nm), RY586 can be used as an alternative to PE and we recommend using an optical filter centered near 586-nm (eg, a 586/15-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE. Compared to PE, RY586 is similar in brightness, minimal spillover into Blue detectors, and increased spillover into the 610/20-nm (PE-CF594) detector. Please ensure that your instrument configuration (lasers and optical filters) is appropriate for this dye.
RY586
Yellow-Green 561 nm
564 nm
586 nm
Citations & References
Development References (7)
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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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Burdin N, Peronne C, Banchereau J, Rousset F. Epstein-Barr virus transformation induces B lymphocytes to produce human interleukin 10. J Exp Med. 1993; 177(2):295-304. (Clone-specific: ELISA). View Reference
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Gotlieb WH, Abrams JS, Watson JM, Velu TJ, Berek JS, Martinez-Maza O. Presence of interleukin 10 (IL-10) in the ascites of patients with ovarian and other intra-abdominal cancers. Cytokine. 1992; 4(5):385-390. (Clone-specific: ELISA). View Reference
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Jeffery LE, Burke F, Mura M, et al. 1,25-Dihydroxyvitamin D3 and IL-2 combine to inhibit T cell production of inflammatory cytokines and promote development of regulatory T cells expressing CTLA-4 and FoxP3.. J Immunol. 2009; 183(9):5458-67. (Clone-specific: Flow cytometry). 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
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Yssel H, De Waal Malefyt R, Roncarolo MG, et al. IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J Immunol. 1992; 149(7):2378-2384. (Clone-specific: ELISA). View Reference
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.
