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Flow cytometric analysis of CD137 (4-1BB) expression on viable stimulated Human peripheral blood lymphocytes. Concanavalin A (Con-A)-stimulated (3 days) peripheral blood mononuclear cells (PBMC) were stained with either BD Horizon™ RB705 Mouse IgG1, κ Isotype Control (Cat. No. 570261; dashed line histogram) or BD Horizon™ RB705 Mouse Anti-Human CD137 (4-1BB) antibody (Cat. No. 570577/570665; solid line histogram). DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Solution (Cat. No. 564907) was added to cells right before analysis. The fluorescence histogram showing the expression of CD137 (4-1BB) [or Ig Isotype control staining] was derived from gated events with the forward and side light-scatter characteristics of viable (DAPI-negative) activated lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ Software.
BD Horizon™ RB705 Mouse Anti-Human CD137 (4-1BB)
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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
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- 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.
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- 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.
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Companion Products
The 4B4-1 monoclonal antibody specifically binds to CD137 which is also known as 4-1BB, and ILA (induced by lymphocyte activation). CD137 is a type I transmembrane glycoprotein that belongs to the TNF/NGF receptor family. It is encoded by TNFRSF9 (tumor necrosis factor receptor superfamily, member 9). CD137 is expressed on activated T cells, B cells, monocytes, and follicular dendritic cells. CD137 plays roles in the costimulation, differentiation and survival of T cells and B cells.
Development References (6)
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Garni-Wagner BA, Lee ZH, Kim YJ, Wilde C, Kang CY, Kwon BS. 4-1BB is expressed on CD45RAhiROhi transitional T cell in humans. Cell Immunol. 1996; 169(1):91-98. (Immunogen: ELISA, Flow cytometry, Immunoprecipitation). View Reference
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Goodwin, RG. CDw137 (4-1BB) Workshop Panel report. In: Kishimoto T. Tadamitsu Kishimoto .. et al., ed. Leucocyte typing VI : white cell differentiation antigens : proceedings of the sixth international workshop and conference held in Kobe, Japan, 10-14 November 1996. New York: Garland Pub.; 1997:883-884.
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Hurtado JC, Kim SH, Pollok KE, Lee ZH, Kwon BS. Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28. J Immunol. 1995; 155(7):3360-3367. (Biology). View Reference
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Kim YJ, Broxmeyer HE. Therapeutic potential of 4-1BB (CD137) as a regulator for effector CD8(+) T cells. J Hematother Stem Cell Res. 2001; 10(4):441-449. (Biology). View Reference
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Zacharakis N, Chinnasamy H, Black M, et al. Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer.. Nat Med. 2018; 24(6):724-730. (Clone-specific: Flow cytometry). View Reference
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Zhou Z, Kim S, Hurtado J, et al. Characterization of human homologue of 4-1BB and its ligand. Immunol Lett. 1995; 45(1-2):67-73. (Biology). View Reference
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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.