BD Pharmingen™ Alexa Fluor™ 488 Biosimilar Anti-Human TNF
Clone Infliximab297.rMAb (RUO)
Two-color flow cytometric analysis of TNF (Infliximab Biosimilar) in 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 fixed and permeabilized cells were then stained in BD Perm/Wash™ Buffer with either Alexa Fluor™ 488 IgG1, κ N297A Human Isotype Control (Cat. No. 570390; Left Plot) or Alexa Fluor™ 488 Biosimilar Anti-Human TNF antibody (Cat. No. 570954/571025; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus side light-scatter (SSC-A) signals 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 BD LSRFortessa™ Cell Analyzer System and FlowJo™ Software.
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Two-color flow cytometric analysis of TNF (Infliximab Biosimilar) in 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 fixed and permeabilized cells were then stained in BD Perm/Wash™ Buffer with either Alexa Fluor™ 488 IgG1, κ N297A Human Isotype Control (Cat. No. 570390; Left Plot) or Alexa Fluor™ 488 Biosimilar Anti-Human TNF antibody (Cat. No. 570954/571025; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus side light-scatter (SSC-A) signals 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 BD LSRFortessa™ Cell Analyzer System and FlowJo™ Software.
Preparation And Storage
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.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- 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).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- An isotype control should be used at the same concentration as the antibody of interest.
- This product is provided under an intellectual property license between Life Technologies Corporation and BD Businesses. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for any other use, contact Life Technologies Corporation, Cell Analysis Business Unit Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA, Tel: (541) 465-8300. Fax: (541) 335-0504.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Alexa Fluor™ is a trademark of Life Technologies Corporation.
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
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The Infliximab N297A Biosimilar.rMAb is a research grade recombinant Human IgG1, kappa monoclonal antibody that specifically recognizes Human Tumor Necrosis Factor (TNF) similarly to therapeutic Infliximab. Infliximab N297A Biosimilar.rMAb was engineered to code for a replacement of asparagine with alanine at position 297 (N297A) of the Human IgG1 heavy chain to reduce potential nonspecific staining caused by Fc receptor binding. Infliximab is a chimeric recombinant Human IgG1 Anti-Human TNF monoclonal antibody that is used to treat a wide variety of inflammatory conditions such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, Bechet's disease, psoriasis, septic shock, cachexia, and cancer. Therapeutic Infliximab specifically binds to soluble and transmembrane TNF and neutralizes its biological activity by blocking the binding to its cell surface receptors. It may also bind to transmembrane TNF on cells that produce it and mediate both antibody-dependent (ADCC) and complement-dependent (CDC) cytotoxicity. Infliximab does not bind to or inactivate lymphotoxin (Tumor necrosis factor-beta; TNF-ß). TNF, also known as TNF alpha (TNF-a) or Cachectin, is an ~26 kDa type II transmembrane protein that is encoded by TNF. TNF is a multifunctional cytokine that plays roles in inflammation, immune system development, innate and adaptive immune responses, apoptosis, lipid metabolism, and necrosis of some tumor cells. TNF is variably produced by a wide variety of activated leucocytes, epithelial cells, endothelial cells, and tumor cells. This mediator forms noncovalently bound homotrimers that are expressed on the cell surface. Membrane TNF is enzymatically cleaved from the cell surface by tumor necrosis factor alpha converting enzyme (TACE), also known as ADAM17 and CD156b, into a soluble biologically active trimer of the TNF extracellular domains. TNF mediates its biological activities through binding to cell surface TNF Receptor Type 1 (TNFR1, CD120a) and Type 2 (TNFR2, CD120b), which are widely expressed on normal hemopoietic and nonhemopoietic cells as well as tumor cells.
The Infliximab N297A Biosimilar.rMAb is intended for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals.
Development References (3)
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Knight DM, Trinh H, Le J, et al. Construction and initial characterization of a mouse-human chimeric anti-TNF antibody.. Mol Immunol. 1993; 30(16):1443-53. (Immunogen: Blocking, ELISA, Radioimmunoassay). View Reference
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Mitoma H, Horiuchi T, Tsukamoto H, Ueda N. Molecular mechanisms of action of anti-TNF-α agents - Comparison among therapeutic TNF-α antagonists.. Cytokine. 2018; 101:56-63. (Biology). View Reference
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Scallon BJ, Moore MA, Trinh H, Knight DM, Ghrayeb J. Chimeric anti-TNF-alpha monoclonal antibody cA2 binds recombinant transmembrane TNF-alpha and activates immune effector functions.. Cytokine. 1995; 7(3):251-9. (Clone-specific: Blocking, Cytotoxicity, Flow cytometry, Inhibition, Radioimmunoassay). 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.
Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.