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) 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 restimulated for 5 hr with Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), and fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (Cat. No. 554722). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with either BD Horizon™ BV650 Rat IgG1 κ Isotype Control (Cat. No. 563848; Left Panel) or BD Horizon™ BV650 Mouse Anti-Human IL-10 and Viral IL-10 antibody (Cat. No. 564051; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot plots show the correlated expression patterns of IL-10 (or Ig Isotype control staining) versus autofluorescence for gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
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) 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 restimulated for 5 hr with Phorbol 12-Myristate 13-Acetate (Sigma, Cat. No. P-8139; 50 ng/ml) and Ionomycin (Sigma, Cat. No. I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), and fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (Cat. No. 554722). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with either BD Horizon™ BV650 Rat IgG1 κ Isotype Control (Cat. No. 563848; Left Panel) or BD Horizon™ BV650 Mouse Anti-Human IL-10 and Viral IL-10 antibody (Cat. No. 564051; Right Panel) using BD Biosciences Intracellular Cytokine Staining protocol. Two-color flow cytometric dot plots show the correlated expression patterns of IL-10 (or Ig Isotype control staining) versus autofluorescence for gated events with the forward and side light-scatter characteristics of intact stimulated lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
製品詳細
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
AB_2738565
Aqueous buffered solution containing BSA and ≤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 with BD Horizon™ BV650 under optimum conditions, and unconjugated antibody and free BD Horizon™ BV650 were removed.
Product Notices
- 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).
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Brilliant Violet™ 650 is a trademark of Sirigen.
- 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.
関連製品
Stain Buffer (FBS) RUO
サイズ
500 mL
カタログ番号
554656
Fixation and Permeabilization Solution RUO
サイズ
125 mL
カタログ番号
554722
Perm/Wash Buffer RUO
サイズ
100 mL
カタログ番号
554723
Protein Transport Inhibitor (Containing Monensin) RUO
サイズ
0.7 mL
カタログ番号
554724
BV650 Rat IgG1, κ Isotype Control RUO
サイズ
50 µg
カタログ番号
563848
Purified NA/LE Mouse Anti-Human CD3 RUO
サイズ
0.5 mg
カタログ番号
555329
564051 Rev. 1
抗体の詳細
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.
The antibody was conjugated to BD Horizon™ BV650 which is part of the BD Horizon™ Brilliant Violet™ family of dyes. This dye is a tandem fluorochrome of BD Horizon™ BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 650-nm. BD Horizon™ BV650 can be excited by the violet laser and detected in a filter used to detect APC-like dyes (eg, 660/20-nm filter). Due to the excitation and emission characteristics of the acceptor dye, there will be spillover into the APC and Alexa Fluor® 700 detectors. However, the spillover can be corrected through compensation as with any other dye combination.
564051 Rev. 1
フォーマットの詳細
BV650
The BD Horizon Brilliant Violet™ 650 (BV650) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This tandem fluorochrome is comprised of a BV421 donor with an excitation maximum (Ex Max) of 406-nm and an acceptor dye with an emission maximum (Em Max) at 649-nm. BV650, driven by BD innovation, is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 650-nm (e.g., a 660/20-nm bandpass filter). The acceptor dye can be excited by the Red (628–640-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
BV650
Violet 405 nm
406 nm
649 nm
564051 Rev.1
引用＆参考文献
Development References (6)
-
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
-
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
-
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
-
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
-
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
-
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
564051 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.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
