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Multicolor flow cytometric analysis of (CD370) Clec9A expression on mouse splenocytes. Mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with PE Rat Anti-Mouse CD4 (Cat. No. 553048/553049/561837), FITC Rat Anti-Mouse CD8a (Cat. No. 553031/553030/561966), APC Hamster Anti-Mouse CD11c (Cat. No. 550261/561119) antibodies and either BD Horizon™ BV421 Rat IgG2a κ Isotype Control (Cat. No. 562602; Left Panel) or BD Horizon BV421 Rat Anti-Mouse CD370 (Clec9A) antibody (Cat. No. 564271; Right Panel). Two color flow cytometric contour plots showing the correlated expression of CD11c and Clec9A (or Ig Isotype control staining) were derived from CD4- CD8a+ gated events with the forward and side light-scatter characteristics of viable cells. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.


BD Horizon™ BV421 Rat Anti-Mouse CD370 (Clec9A)

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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
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- 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.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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The 10B4 monoclonal antibody specifically binds to mouse Clec9A. Mouse Clec9A (C-type lectin domain family member 9A) is also known as DNGR1 (Dendritic cell natural killer lectin group receptor 1). It is a type II membrane protein with a single extracellular C-type lectin domain. Clec9A is a dendritic cell subtype-restricted C-type lectin-like receptor. Clec9A is selectively expressed on plasmacytoid dendritic cells and CD8+ myeloid dendritic cells. Clec9A reportedly serves as a receptor for necrotic cells. It can mediate the cross-presentation of dead-cell associated antigens in a Syk-dependent manner.
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 (3)
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Caminschi L, Proietto AL, Ahmet F, et al. The dendritic cell subtype restricted C-type lectin Clec9A is a target for vaccine enhancement. Blood. 2008; 112(8):3264-3273. (Immunogen: Flow cytometry, Functional assay, Immunohistochemistry, In vivo exacerbation). View Reference
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Huysamen C, Willment JA, Dennehy KM, Brown GD. CLEC9A is a novel activation C-type lectin-like receptor expressed on BDCA3+ dendritic cells and a subset of monocytes. J Biol Chem. 2008; 283(24):16693-16701. (Biology). View Reference
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Sancho D, Joffre OP, Keller AM, et al. Identification of a dendritic cell receptor that couples sensing of necrosis to immunity. Nature. 2009; 458(7240):899-903. (Biology). View Reference
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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.