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      Orange Nitric Oxide Probe

      BD Pharmingen™ Orange Nitric Oxide Probe

      (RUO)
      Orange Nitric Oxide Probe
      Figure 1. Reactivity of BD Pharmingen™ Orange Nitric Oxide Probe to a Variety of Reactive Nitrogen and Oxygen Species. BD Pharmingen™ Orange Nitric Oxide Probe was dissolved in PBS and exposed to various reactive nitrogen and oxygen species. Probe fluorescence was then collected using a spectrophotometer with 540 nm light excitation and a 585/10 bandpass filter for emission. Orange Nitric Oxide Probe is forty- to one thousand-fold more sensitive to nitric oxide than other reactive nitrogen and oxygen species.
      Orange Nitric Oxide Probe
      Figure 2. BD Pharmingen™ Orange Nitric Oxide Probe for the flow cytometric analysis of Nitric Oxide levels generated in Jurkat cells using DEA NONOate. Cells from the human Jurkat (Acute T cell leukemia, ATCC TIB-152) cell line in 1× DPBS were preincubated with (red line histograms) or without (blue line histograms) BD Pharmingen™ Orange Nitric Oxide Probe for 30 minutes at 37°C and then treated with (solid histogram lines) or without (dashed histogram lines) 100 µM Diethylamine NONOate (Sigma Aldrich, Cat. No. D5431) for 30 minutes at 37°C. Cells were washed once and then analyzed by flow cytometry using 488 nm excitation (Left Panel) or 561 nm (Right Panel) laser excitation and a 575/25 nm bandpass filter to collect fluorescent light emissions. Treatment with the nitric oxide donor Diethylamine NONOate resulted in increased fluorescence of nitric oxide-producing cells due to reacted Orange Nitric Oxide Probe. The histograms were derived from gated events with the forward and side light-scattering characteristics of intact Jurkat cells. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.
      Orange Nitric Oxide Probe Orange Nitric Oxide Probe
      Figure 1. Reactivity of BD Pharmingen™ Orange Nitric Oxide Probe to a Variety of Reactive Nitrogen and Oxygen Species. BD Pharmingen™ Orange Nitric Oxide Probe was dissolved in PBS and exposed to various reactive nitrogen and oxygen species. Probe fluorescence was then collected using a spectrophotometer with 540 nm light excitation and a 585/10 bandpass filter for emission. Orange Nitric Oxide Probe is forty- to one thousand-fold more sensitive to nitric oxide than other reactive nitrogen and oxygen species.
      Figure 2. BD Pharmingen™ Orange Nitric Oxide Probe for the flow cytometric analysis of Nitric Oxide levels generated in Jurkat cells using DEA NONOate. Cells from the human Jurkat (Acute T cell leukemia, ATCC TIB-152) cell line in 1× DPBS were preincubated with (red line histograms) or without (blue line histograms) BD Pharmingen™ Orange Nitric Oxide Probe for 30 minutes at 37°C and then treated with (solid histogram lines) or without (dashed histogram lines) 100 µM Diethylamine NONOate (Sigma Aldrich, Cat. No. D5431) for 30 minutes at 37°C. Cells were washed once and then analyzed by flow cytometry using 488 nm excitation (Left Panel) or 561 nm (Right Panel) laser excitation and a 575/25 nm bandpass filter to collect fluorescent light emissions. Treatment with the nitric oxide donor Diethylamine NONOate resulted in increased fluorescence of nitric oxide-producing cells due to reacted Orange Nitric Oxide Probe. The histograms were derived from gated events with the forward and side light-scattering characteristics of intact Jurkat cells. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.
      Product Details
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      BD Pharmingen™
      Flow cytometry (Tested During Development)
      AB_2869737
      RUO


      Preparation And Storage

      Store undiluted at -20°C and protected from prolonged exposure to light. Small aliquots of the stock solution may be stored frozen, undiluted, and protected from light at -20°C.

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      Recommended Assay Procedures

      Cytometry Requirements

      Yellow-green or blue laser-equipped flow cytometers (eg, BD FACSCanto™ II, BD LSRFortessa™, BD LSR™ II, or BD Accuri™ C6) can be used. This dye can be read out of filters commonly used for PE (eg, 575/26- or 582/15-nm filter). Fluorescence compensation is best achieved using stained and unstained samples of the cells of interest. When designing multicolor panels, please be aware of spillover into the BD Horizon™ BV605 and BV650 channels. Panels should be optimized to take this spillover into account.

      Procedure

      Staining of Live Cells for Analysis by Flow Cytometry

      1.        Count cells to determine cell density. If necessary, adjust cell density to 1-10 × 10e6 cells/mL in fresh, pre-warmed 1× DPBS, cell culture media, or desired stain buffer.

      2.        Add 1 μL BD Pharmingen™ Orange Nitric Oxide Probe per 500 μL cell suspension.

      a.        We recommend titrating the dye for optimal performance, as different cell types, incubation periods, or culture conditions can result in variability in staining.

      3.        Without washing, treat cells as desired at 37°C for desired period of time to generate nitric oxide.

      a.        If cytotoxicity is observed, cells may be pre-incubated with BD Pharmingen™ Orange Nitric Oxide Probe for 30-60 minutes at 37°C, washed once to remove excess dye, and then exposed to treatment conditions.

      b.        A positive control may be generated by pre-incubating cells with BD Pharmingen™ Orange Nitric Oxide Probe for 30-60 minutes at 37°C and then treating cells with 1 mM DEA NONOate (Sigma Cat. No. D5431) for 30-60 minutes at 37°C.

      4.        Wash cells once to remove treatment compounds.

      5.        Proceed to analysis by flow cytometry.

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      Product Notices

      1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
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      566065 Rev. 1
      Antibody Details
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      Nitric oxide is a signaling molecule involved in nervous system signal transduction, blood vessel tone in the cardiovascular system, and host defense in immunological systems. In these systems, nitric oxide production is predominantly catalyzed by the enzymes nNOS, eNOS, and iNOS, respectively. Because nitric oxide is a free radical, its overproduction is implicated in cytotoxic proinflammatory disorders.

      BD Pharmingen™ Orange Nitric Oxide Probe provides an indicator that allows specific measurement of intracellular nitric oxide levels within live cells. In its unreacted form, the probe has minimal fluorescence and freely crosses the cell membrane. Once it reacts with intracellular nitric oxide, the oxidized probe shows increased fluorescence and becomes trapped within the cell. Cells with higher levels of intracellular nitric oxide will therefore display increased fluorescence intensity that can be analyzed by flow cytometry. This probe has been shown to be at least as sensitive as DAF-FM DA.

      The reacted BD Pharmingen™ Orange Nitric Oxide Probe has an excitation maximum of 545 nm and an emission maximum of 576 nm. It is excited by the blue or yellow-green lasers.

      566065 Rev. 1
      Citations & References
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      Development References (8)

      1. Balaguer S, Diaz L, Gomes A, et al. Real-time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no-wash, no-lyse kinetic method.. Cytometry B Clin Cytom. 2015. (Methodology). View Reference
      2. Balcerczyk A, Soszynski M, Bartosz G. On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide.. Free Radic Biol Med. 2005; 39(3):327-35. (Methodology). View Reference
      3. Freitas M, Lima JL, Fernandes E. Optical probes for detection and quantification of neutrophils' oxidative burst. A review.. Anal Chim Acta. 2009; 649(1):8-23. (Methodology). View Reference
      4. Itoh Y, Ma FH, Hoshi H, et al. Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry.. Anal Biochem. 2000; 287(2):203-9. (Methodology). View Reference
      5. Ku CJ, Karunarathne W, Kenyon S, Root P, Spence D. Fluorescence determination of nitric oxide production in stimulated and activated platelets.. Anal Chem. 2007; 79(6):2421-6. (Methodology). View Reference
      6. McQuade LE, Lippard SJ. Fluorescent probes to investigate nitric oxide and other reactive nitrogen species in biology (truncated form: fluorescent probes of reactive nitrogen species).. Curr Opin Chem Biol. 2010; 14(1):43-9. (Methodology). View Reference
      7. O'Brien GC, Wang JH, Redmond HP. Bacterial lipoprotein induces resistance to Gram-negative sepsis in TLR4-deficient mice via enhanced bacterial clearance.. J Immunol. 2005; 174(2):1020-6. (Methodology). View Reference
      8. Pye D, Palomero J, Kabayo T, Jackson MJ. Real-time measurement of nitric oxide in single mature mouse skeletal muscle fibres during contractions.. J Physiol (Lond). 2007; 581(Pt 1):309-18. (Methodology). View Reference
      View All (8) View Less
      566065 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.

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