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Flow cytometric analysis of CD16 expression on human peripheral blood lymphocytes. Peripheral blood cells from human were stained with PE-Cy™5 Mouse Anti-Human CD16 antibody (Cat. No. 555408/561725; solid line histogram) or PE-Cy™5 Mouse IgG1, κ Isotype Control (Cat. No. 555750; dashed line histogram). The erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry was performed using a BD FACScan™ Flow Cytometer System.
BD Pharmingen™ PE-Cy™5 Mouse Anti-Human CD16
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Product Notices
- 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.
- 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.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- PE-Cy5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the PE-Cy5 tandem fluorochrome, extra care must be taken when using dual-laser cytometers which may directly excite both PE and Cy5™.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- PE-Cy5 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by the 488 nm light of an Argon ion laser and serves as an energy donor, coupled to the cyanine dye Cy5, which acts as an energy acceptor and fluoresces at 670 nm. BD Biosciences Pharmingen has maximized the fluorochrome energy transfer in PE-Cy5, thus maximizing its fluorescence emission intensity, minimizing residual emission from PE, and minimizing lot-to-lot variation.
- PE-Cy5 tandem fluorochromes have been reported to bind some classes of human macrophages and granulocytes via Fc receptors, and PE has been reported to bind to mouse B lymphocytes via Fc receptors. Preincubation of mouse leukocytes with Mouse BD Fc Block™ purified anti-mouse CD16/CD32 mAb 2.4G2 can reduce the non-specific binding of PE-Cy5-conjugated reagents to mouse B cells. However, PE-Cy5 conjugated reagents should not be used to stain splenocytes of SJL, NOD, and MRL mice as B lymphocytes and/or other leukocytes have been reported to non-specifically stain regardless of the use of Mouse BD Fc Block™ (the CD72c complex has been implicated for PE-Cy5 binding in these strains). Reagents conjugated to PE, PerCP, PerCP-Cy5.5, APC, and APC-Cy7 tandem fluorochrome can be used on leukocytes from these mouse strains.
- Cy is a trademark of GE Healthcare.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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.
This clone also cross-reacts with a subset of peripheral blood lymphocytes and monocytes, but not granulocytes, of baboon and both rhesus and cynomologus macaque monkeys. Multi-color 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.
Development References (5)
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Barclay NA, Brown MH, Birkeland ML, et al, ed. The Leukocyte Antigen FactsBook. San Diego, CA: Academic Press; 1997.
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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
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Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
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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
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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
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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.