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Two-color flow cytometric analysis of CD16/32 expression on mouse splenocytes. C57BL/6 mouse splenic leucocytes were stained with Alexa Fluor® 647 Rat Anti-Mouse CD3 (Cat. No. 557869) and either BD Horizon™ BV421 Rat IgG2b, κ Isotype Control (Cat. No. 562603; Left Plot) or BD OptiBuild™ BV421 Rat Anti-Mouse CD16/CD32 (Cat. No. 747952; Right Plot) at 0.5 μg/test. The pseudocolor dot plots showing the correlated expression of CD16/CD32 (or Ig Isotype control staining) versus CD3 were derived from gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software. The above is qualification data only and does not represent a specific OptiBuild™ lot.
BD OptiBuild™ BV421 Rat Anti-Mouse CD16/CD32
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
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.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
Product Notices
- The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
- Researchers should determine the optimal concentration of this reagent for their individual applications.
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
- BD Horizon Brilliant Violet 421 is covered by one or more of the following US patents: 8,158,444; 8,362,193; 8,575,303; 8,354,239.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
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Companion Products
The 190909 antibody specifically recognizes a common epitope on the extracellular domains of CD16 (also known as, Fc gamma RIII) and CD32 (Fc gamma RII). CD16 and CD32 serve as low affinity receptors for mouse IgG Fc constant regions and play roles in the activation or inhibition of phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), degranulation, and B cell proliferation. These receptors are variably expressed on B lymphocytes, natural killer (NK) cells, monocytes, macrophages, dendritic cells, Kupffer cells, granulocytes, mast cells, immature thymocytes, and some activated mature T lymphocytes. CD16 and CD32 are single-pass type I transmembrane glycoproteins with two extracellular Ig-like domains and belong to the Ig superfamily. Ligand-bound CD16 can associate with two disulfide-linked FcR-gamma subunits (FcεRIγ) that contain cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) which deliver activating signals intracellularly. CD32 has an intracellular domain with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and functions as an inhibitory receptor.
The antibody was conjugated to BD Horizon BV421 which is part of the BD Horizon Brilliant™ Violet family of dyes. With an Ex Max near 407 nm and Em Max near 421 nm, BD Horizon BV421 can be excited by the violet laser (405 nm) and detected with a 450/50 nm filter. BD Horizon BV421 conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific BlueTM conjugates. Due to nearly identical excitation and emission properties but different spillover characteristics, BD Horizon BV421, Pacific Blue, and BD Horizon V450 cannot be used simultaneously.
Development References (2)
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Kool M, Geurtsvankessel C, Muskens F, et al. Facilitated antigen uptake and timed exposure to TLR ligands dictate the antigen-presenting potential of plasmacytoid DCs.. J Leukoc Biol. 2011; 90(6):1177-90. (Clone-specific: Flow cytometry). View Reference
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Nimmerjahn F, Ravetch JV. FcγRs in health and disease. Curr Top Microbiol Immunol. 2011; 350:105-125. (Biology). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.