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V450 Mouse Anti-Human CD16
V450 Mouse Anti-Human CD16

Flow cytometric analysis of BD Horizon™ V450 anti-human CD16 on human lymphocytes. Whole blood was stained with V450 anti-human CD16 (Cat. No.560474/560475/561246; solid line histogram) and compared to whole blood stained with a V450 mouse IgG1, κ isotype control (Cat. No. 560373; dashed line histogram). Lymphocytes were selected by light scatter profile. Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Flow cytometric analysis of BD Horizon™ V450 anti-human CD16 on human lymphocytes. Whole blood was stained with V450 anti-human CD16 (Cat. No.560474/560475/561246; solid line histogram) and compared to whole blood stained with a V450 mouse IgG1, κ isotype control (Cat. No. 560373; dashed line histogram). Lymphocytes were selected by light scatter profile. Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Product Details
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BD Horizon™
CD16;CD16A;FCGR3A;FcγRIIIA;FcRIIIa;CD16B;FCGR3B;FcγRIIIB;FcRIIIb
Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Mouse BALB/c x DBA/2, also known as CD2F1 or CDF1 IgG1, κ
Human polymorphonuclear leukocytes
Flow cytometry (Routinely Tested)
5 µl
IV N409; V MR5, NK80
AB_1645561
Aqueous buffered solution containing protein stabilizer and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. 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.

Recommended Assay Procedures

BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation).  When fluorochrome conjugated antibodies are bound to 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 CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.

Product Notices

  1. 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).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. BD Horizon V450 has a maximum absorption of 406 nm and maximum emission of 450 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. Pacific Blue™ is a trademark of Life Technologies Corporation.
  8. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560475 Rev. 3
Antibody Details
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3G8

The 3G8 monoclonal antibody specifically recognizes CD16a and CD16b, low affinity receptors for the Fc region of IgG. CD16a is ~50-65 kDa type I transmembrane glycoprotein that is encoded by FCGR3A (Fc fragment of IgG receptor IIIa) which belongs to the immunoglobulin superfamily. CD16a is also known as Fc-gamma RIII-alpha (Fc-gamma RIIIa or FcγRIIIA) or FcRIIIa and is expressed on natural killer cells, activated monocytes, macrophages, γδ T cells, immature thymocytes, and mast cells. CD16a binds immune-complexed or aggregated IgG and associates with CD247/TCRζ in NK cells and FcεRIγ chains in phagocytes and mast cells to transduce intracellular signals. CD16a functions in antibody-dependent cellular cytotoxicity (ADCC) and other antibody-dependent responses including phagocytosis, cytokine production or mediator release. CD16b is a ~48 kDa glycophosyl-phosphatidylinositol (GPI)-linked form that is encoded by FCGR3B (Fc fragment of IgG receptor IIIb). CD16b is also known as Fc-gamma RIII-beta (Fc-gamma RIIIb or FcγRIIIB) or FcRIIIb and is expressed on neutrophils and activated eosinophils. The extracellular region of CD16b is highly homologous to CD16a. CD16b also serves as a receptor for the Fc region of IgG and can bind immune-complexed or aggregated IgG and may be involved in neutrophil adhesion.

       The 3G8 antibody also crossreacts with a subset of peripheral blood lymphocytes and monocytes, but not granulocytes, of baboon, rhesus, and cynomolgus monkeys. Multicolor analysis reveals that the distribution on lymphocytes is similar to that found in human studies with the majority of CD16-positive lymphocytes being both CD3 and CD20 negative.

The antibody is conjugated to BD Horizon V450, which has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser Ex max of 406 nm and has an Em Max at 450 nm. Conjugates with BD Horizon V450 can be used in place of Pacific Blue™ conjugates.

560475 Rev. 3
Format Details
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V450
BD Horizon™ V450 Dye is part of the BD Horizon™ violet family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 405-nm and an emission maximum (Em Max) at 450-nm. BD Horizon™ V450, driven by BD innovation, is designed to be excited by the violet laser (405 nm) and detected using an optical filter centered near 450-nm (e.g., a 450/50-nm bandpass filter). The dye can be excited by the UV (355-nm) laser resulting in cross-laser excitation and spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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V450
Violet 405 nm
405 nm
450 nm
560475 Rev.3
Citations & References
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Development References (10)

  1. Barclay NA, Brown MH, Birkeland ML, et al, ed. The Leukocyte Antigen FactsBook. San Diego, CA: Academic Press; 1997.
  2. Fleit HB, Wright SD, Durie CJ, Valinsky JE, Unkeless JC. Ontogeny of Fc receptors and complement receptor (CR3) during human myeloid differentiation. J Clin Invest. 1984; 73(2):516-525. (Clone-specific: Flow cytometry, Fluorescence microscopy, Immunofluorescence, Immunoprecipitation, Radioimmunoassay). View Reference
  3. Fleit HB, Wright SD, Unkeless JC. Human neutrophil Fc gamma receptor distribution and structure. Proc Natl Acad Sci U S A. 1982; 79(10):3275-3279. (Biology). View Reference
  4. Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
  5. Perussia B, Trinchieri G, Jackson A, et al. The Fc receptor for IgG on human natural killer cells: phenotypic, functional, and comparative studies with monoclonal antibodies. J Immunol. 1984; 133(1):180-189. (Clone-specific: Flow cytometry, Functional assay, Inhibition). View Reference
  6. Schmidt RE. Non-lineage/natural killer section report: new and previously defined clusters. In: Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:517-542.
  7. Stroncek DF, Skubitz KM, Plachta LB, et al. Alloimmune neonatal neutropenia due to an antibody to the neutrophil Fc-gamma receptor III with maternal deficiency of CD16 antigen. Blood. 1991; 77(7):1572-1580. (Biology). View Reference
  8. Vossebeld PJ, Homburg CH, Roos D, Verhoeven AJ. The anti-Fc gamma RIII mAb 3G8 induces neutrophil activation via a cooperative actin of Fc gamma RIIIb and Fc gamma RIIa. Int J Biochem Cell Biol. 1997; 29(3):465-473. (Clone-specific: Activation, Functional assay). View Reference
  9. Wirthmueller U, Kurosaki T, Murakami MS, Ravetch JV. Signal transduction by Fc gamma RIII (CD16) is mediated through the gamma chain. J Exp Med. 1992; 175(5):1381-1390. (Biology). View Reference
  10. Zola H. Leukocyte and stromal cell molecules : the CD markers. Hoboken, N.J.: Wiley-Liss; 2007.
View All (10) View Less
560475 Rev. 3

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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


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.