Two-color flow cytometric analysis of TNF expression by stimulated Human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 μg/ml final concentration) 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 with BD Cytofix™ Fixation Buffer (Cat. No. 554655) followed by washing and permeabilization using BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained in BD Perm/Wash™ Buffer with BD Horizon™ BV421 Mouse Anti-Human CD3 antibody (Cat. No. 562426) and with either BD Horizon™ RY610 Mouse IgG1, κ Isotype Control (Cat. No. 571151; Left Plot) or BD Horizon™ RY610 Mouse Anti-Human TNF antibody (Cat. No. 571267/571327; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus CD3 was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ Software.
Two-color flow cytometric analysis of TNF expression by stimulated Human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 μg/ml final concentration) 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 with BD Cytofix™ Fixation Buffer (Cat. No. 554655) followed by washing and permeabilization using BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained in BD Perm/Wash™ Buffer with BD Horizon™ BV421 Mouse Anti-Human CD3 antibody (Cat. No. 562426) and with either BD Horizon™ RY610 Mouse IgG1, κ Isotype Control (Cat. No. 571151; Left Plot) or BD Horizon™ RY610 Mouse Anti-Human TNF antibody (Cat. No. 571267/571327; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus CD3 was derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ Software.
Product Details
BD Horizon™
Tumor necrosis factor alpha; TNF-a; TNF-α; TNFSF2; Cachectin
Human (QC Testing), Rhesus,Cynomolgus,Baboon (Tested in Development)
Mouse IgG1, κ
Recombinant Human TNF
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl/test
7124
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 to the dye under optimum conditions and unreacted dye was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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.
- 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.
- Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
- 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.
- 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.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- CF™ is a trademark of Biotium, Inc.
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
RY610 Mouse IgG1, κ Isotype Control RUO
Size
0.1 mg
Cat No.
571151
Protein Transport Inhibitor (Containing Monensin) RUO
Size
0.7 mL
Cat No.
554724
571327 Rev. 1
Antibody Details
MAb11
The MAb11 monoclonal antibody specifically binds to human tumor necrosis factor (TNF, also known as TNF-α) protein. TNF is an efficient juxtacrine, paracrine and endocrine mediator of inflammatory and immune functions. It regulates the growth and differentiation of a variety of cell types. TNF is cytotoxic for transformed cells when in conjunction with IFN-γ. It is secreted by activated monocytes/macrophages and other cells such as B cells, T cells and fibroblasts. The immunogen used to generate the MAb11 hybridoma was recombinant human TNF. The MAb11 antibody has been reported to crossreact with Rhesus Macaque TNF.
571327 Rev. 1
Format Details
RY610
The BD Horizon RealYellow™ 610 (RY610) Dye is part of the BD® family of yellow-green dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 557-nm and an emission maximum (Em Max) at 610-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RY610 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), RY610 can be used as an alternative to PE-CF594 and we recommend using an optical filter centered near 610-nm (eg, a 610/20-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE-CF594.
RY610
Yellow-Green 561 nm
557 nm
610 nm
571327 Rev.1
Citations & References
View product citations for antibody "571327" on CiteAb
Development References (12)
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Andersen H, Rossio JL, Coalter V, et al. Characterization of rhesus macaque natural killer activity against a rhesus-derived target cell line at the single-cell level.. Cell Immunol. 231(1-2):85-95. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Castro I, Giret TM, Magnani DM, et al. Cellular Immune Responses against Simian T-Lymphotropic Virus Type 1 Target Tax in Infected Baboons.. J Virol. 2016; 90(11):5280-5291. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Danis VA, Franic GM, Rathjen DA, Brooks PM. Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-6 on the production of immunoreactive IL-1 and TNF-alpha by human monocytes. Clin Exp Immunol. 1991; 85(1):143-150. (Clone-specific: ELISA). View Reference
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Engele M, Stössel E, Castiglione K, et al. Induction of TNF in human alveolar macrophages as a potential evasion mechanism of virulent Mycobacterium tuberculosis.. J Immunol. 2002; 168(3):1328-37. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Foulds KE, Donaldson M, Roederer M. OMIP-005: Quality and phenotype of antigen-responsive rhesus macaque T cells.. Cytometry A. 2012; 81(5):360-1. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Geisbert TW, Bailey M, Geisbert JB, et al. Vector choice determines immunogenicity and potency of genetic vaccines against Angola Marburg virus in nonhuman primates.. J Virol. 2010; 84(19):10386-94. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Jason J, Larned J. Single-cell cytokine profiles in normal humans: comparison of flow cytometric reagents and stimulation protocols. J Immunol Methods. 1997; 207(1):13-22. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Lecoeur H, Ledru E, Gougeon ML. A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes.. J Immunol Methods. 1998; 217(1-2):11-26. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Mascher B, Schlenke P, Seyfarth M. Expression and kinetics of cytokines determined by intracellular staining using flow cytometry. J Immunol Methods. 1999; 223(1):115-121. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Moris P, Bellanger A, Ofori-Anyinam O, Jongert E, Yarzabal Rodriguez JP, Janssens M. Whole blood can be used as an alternative to isolated peripheral blood mononuclear cells to measure in vitro specific T-cell responses in human samples.. J Immunol Methods. 2021; 492:112940. (Clone-specific: Flow cytometry, Intracellular Staining/Flow Cytometry). View Reference
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Pitcher CJ, Hagen SI, Walker JM, et al. Development and homeostasis of T cell memory in rhesus macaque. J Immunol. 2002; 168(1):29-43. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Rathjen DA, Cowan K, Furphy LJ, Aston R. Antigenic structure of human tumour necrosis factor: recognition of distinct regions of TNF alpha by different tumour cell receptors. Mol Immunol. 1991; 28(1-2):79-86. (Immunogen: ELISA). View Reference
571327 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.