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      RB613 Mouse Anti-Human NF-κB p65 (pS529)
      RB613 Mouse Anti-Human NF-κB p65 (pS529)
      Flow cytometric analysis of NF-κB p65 (pS529) expression by TNF-treated HeLa S3 cells. Cultured cells from the Human HeLa S3 (Cervical adenocarcinoma, ATCC® CCL-2.2™) cell line were starved overnight in serum-free RPMI medium. The cells were harvested and washed with Dulbecco's Phosphate Buffered Saline. They were then either left untreated (dashed line histogram) or treated (37°C, 10 min) with Recombinant Human TNF protein (20 ng/mL; Cat. No. 554618; solid line histogram) and Calyculin A (50 nM). The cells were fixed (10 min; 37°C) with pre-warmed BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized (30 min on ice) with BD Phosflow™ Perm Buffer III (Cat. No. 558050) and washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656). The cells were then stained with BD Phosflow™ RB613 Mouse Anti-Human NF-κB p65 (pS529) antibody (Cat. No. 571264/571324).  The fluorescence histograms showing NF-κB p65 (pS529) expression were derived from gated events with the forward and side light-scatter characteristics of intact HeLa S3 cells. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 Cell Analyzer System and FlowJo™ Software.
      RB613 Mouse Anti-Human NF-κB p65 (pS529) RB613 Mouse Anti-Human NF-κB p65 (pS529)
      Flow cytometric analysis of NF-κB p65 (pS529) expression by TNF-treated HeLa S3 cells. Cultured cells from the Human HeLa S3 (Cervical adenocarcinoma, ATCC® CCL-2.2™) cell line were starved overnight in serum-free RPMI medium. The cells were harvested and washed with Dulbecco's Phosphate Buffered Saline. They were then either left untreated (dashed line histogram) or treated (37°C, 10 min) with Recombinant Human TNF protein (20 ng/mL; Cat. No. 554618; solid line histogram) and Calyculin A (50 nM). The cells were fixed (10 min; 37°C) with pre-warmed BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized (30 min on ice) with BD Phosflow™ Perm Buffer III (Cat. No. 558050) and washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656). The cells were then stained with BD Phosflow™ RB613 Mouse Anti-Human NF-κB p65 (pS529) antibody (Cat. No. 571264/571324).  The fluorescence histograms showing NF-κB p65 (pS529) expression were derived from gated events with the forward and side light-scatter characteristics of intact HeLa S3 cells. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 Cell Analyzer System and FlowJo™ Software.
      Product Details
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      BD Phosflow™
      RELA; REL-A; NFKBp65; NFKB3; Transcription factor p65; TF65
      Human (QC Testing)
      Mouse BALB/c IgG2b, κ
      Phosphorylated Human NF-κB p65 Peptide
      Intracellular staining (flow cytometry) (Routinely Tested)
      5 µl/test
      5970
      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.
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      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.

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      Product Notices

      1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      2. 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.
      3. 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.
      4. 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).
      5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      6. An isotype control should be used at the same concentration as the antibody of interest.
      7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
      8. 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.
      9. 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.
      10. Tandem fluorochromes contain both an energy donor and an energy acceptor. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
      11. CF™ is a trademark of Biotium, Inc.
      12. For U.S. patents that may apply, see bd.com/patents.
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      571324 Rev. 1
      Antibody Details
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      K10-895.12.50

      The K10-895.12.50 monoclonal antibody recognizes the phosphorylated serine 529 (pS529) in the transactivation domain of the human NF-κB p65 subunit. Nuclear factor κB (NF-κB) is a ubiquitously expressed transcription factor that regulates the expression of many other genes. It is crucial for cellular responses to a variety of stimuli including stress and microbial pathogens that lead to immunity, inflammation, proliferation, differentiation, survival, apoptosis, and tumorigenesis. The most studied NF-κB complex consists of the p50 (also known as NF-κB1) and p65 (also known as REL-A) subunits, both containing a 300-amino acid region with homology to the Rel proto-oncogene product (RH domain). The RH domain contains motifs for dimerization, nuclear localization, and binding to specific DNA sequences. In addition to the RH domain, the p65 subunit contains the transactivation domain, which is responsible for the interaction with the inhibitor IκB and which contains phosphorylation sites. In most cell types, the p50/p65 heterodimer is located within the cytoplasm complexed to IκB. This complex prevents nuclear translocation and activity of NF-κB. In response to stimuli such as cytokines, LPS, DNA damage, and microbial infections, IκB is phosphorylated at critical residues. This phosphorylation induces dissociation of the IκB/NF-κB complex, allowing the free heterodimeric NF-κB to translocate to the nucleus. Furthermore, optimal activation of NF-κB requires phosphorylation in the transactivation domain of p65. In the nucleus, activated NF-κB dimers bind to the κB sites within promoters and enhancers and function as transcriptional regulators.

      571324 Rev. 1
      Format Details
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      RB613
      The BD Horizon RealBlue™ 613 (RB613) Dye is part of the BD® family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 492-nm and an emission maximum (Em Max) at 613-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RB613 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with reduced excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB613 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 Blue laser (488-nm), it can be used in conjunction with PE-CF594. RB613 is on average brighter than PE-CF594 off the blue laser.
      altImg
      RB613
      Blue 488 nm
      492 nm
      613 nm
      571324 Rev.1
      Citations & References
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      View product citations for antibody "571324" on CiteAb

      Development References (7)

      1. Dominguez-Villar M, Gautron AS, de Marcken M, Keller MJ, Hafler DA. TLR7 induces anergy in human CD4+ T cells. Nat Immunol. 2015; 16(1):118-128. (Clone-specific: Flow cytometry). View Reference
      2. Feasibility study: phospho-specific flow cytometry enabling rapid functional analysis of bone marrow samples from patients with multiple myeloma. Cytometry B Clin Cytom. 2014; 86B:139-144. (Clone-specific: Flow cytometry). View Reference
      3. Mingueneau M, Kreslavsky T, Gray D, . The transcriptional landscape of alphabeta T cell differentiation. Nat Immunol. 2013; 14(6):619-632. (Clone-specific: Flow cytometry). View Reference
      4. Natoli G, Saccani S, Bosisio D, Marazzi I. Interactions of NF-kappaB with chromatin: the art of being at the right place at the right time. Nat Immunol. 2005; 6(5):439-445. (Biology). View Reference
      5. Siebenlist U, Brown K, Claudio E. Control of lymphocyte development by nuclear factor-kappaB. Nat Rev Immunol. 2005; 5:435-445. (Biology). View Reference
      6. Suni MA, Maino VC. Flow cytometric analysis of cell signaling proteins. Methods Mol Biol. 2011; 717:155-169. (Clone-specific: Flow cytometry). View Reference
      7. van de Laar L, van den Bosch A, Boonstra A, et al. PI3K-PKB hyperactivation augments human plasmacytoid dendritic cell development and function. Blood. 2012; 120(25):4982-4991. (Clone-specific: Flow cytometry). View Reference
      View All (7) View Less
      571324 Rev. 1

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      Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

       

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      For Research Use Only. Not for use in diagnostic or therapeutic procedures.