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BV421 Rat Anti-Mouse CD273 (PD-L2)
BV421 Rat Anti-Mouse CD273 (PD-L2)

Multicolor flow cytometric analysis of CD273 (PD-L2) expression on mouse splenic dendritic cells. BALB/c mouse splenic leucocytes were cultured overnight with Recombinant Mouse GM-CSF protein (Cat. No. 554586; 5 ng/ml). The cells were harvested and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with Alexa Fluor™ 700 Hamster Anti-Mouse CD11c antibody (Cat. No. 560583) and either BD Horizon™BV421 Rat IgG2a, κ Isotype Control (Cat. No. 562602; Left Plot) or BD Horizon™BV421 Rat Anti-Mouse CD273 antibody (Cat. No. 567374; Right Plot) at 0.25 μg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of CD273 (PD-L2) [or Ig Isotype control staining] versus CD11c was derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) splenic leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Multicolor flow cytometric analysis of CD273 (PD-L2) expression on mouse splenic dendritic cells. BALB/c mouse splenic leucocytes were cultured overnight with Recombinant Mouse GM-CSF protein (Cat. No. 554586; 5 ng/ml). The cells were harvested and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with Alexa Fluor™ 700 Hamster Anti-Mouse CD11c antibody (Cat. No. 560583) and either BD Horizon™BV421 Rat IgG2a, κ Isotype Control (Cat. No. 562602; Left Plot) or BD Horizon™BV421 Rat Anti-Mouse CD273 antibody (Cat. No. 567374; Right Plot) at 0.25 μg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The bivariate pseudocolor density plot showing the correlated expression of CD273 (PD-L2) [or Ig Isotype control staining] versus CD11c was derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) splenic leucocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.

Product Details
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BD Horizon™
Mouse (QC Testing)
Rat SD, also known as Sprague-Dawley (outbred) IgG2a, κ
Mouse B7-DC-transfected Cell Line
Flow cytometry (Routinely Tested)
0.2 mg/ml
Aqueous buffered solution containing BSA 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 unconjugated antibody and free dye were removed.

Recommended Assay Procedures

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

For optimal and reproducible results, BD Horizon Brilliant™ Stain Buffer should be used anytime BD Horizon Brilliant™ dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant™ Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant™ Stain Buffer is used in in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant™ Stain Buffer for the same length of time as the corresponding multicolor panel. 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

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. 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.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. 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.
  8. 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.
  9. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  10. Pacific Blue™ is a trademark of Life Technologies Corporation.
567374 Rev. 1
Antibody Details
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MIH37

The MIH37 monoclonal antibody specifically recognizes Programmed cell death 1 ligand 2 (PD-L2) which is also known as CD273 and Butyrophilin B7-DC (B7-DC). This regulatory molecule is a 42-kDa type I membrane glycoprotein encoded by Pdcd1lg2 which belongs to the B7 family of the Ig superfamily. CD273 (PD-L2)  is comprised of an extracellular region with an N-terminal IgV-like domain followed by an IgC2-type domain, a transmembrane sequence, and a cytoplasmic tail. Although not detected on resting leucocytes, its expression is upregulated upon activation of macrophages and dendritic cells (DC) by a variety of stimulatory factors including IL-4, IL-13, or GM-CSF. CD273 (PD-L2) serves as a ligand for the coinhibitory receptor, CD279 (PD-1), a receptor that likewise binds to the CD274 (PD-L1) ligand. CD273 (PD-L2) also binds to Repulsive guidance molecule b (RGMb) that is expressed by T cells, macrophages, neutrophils, and DC. RGMb may associate with other receptors that transduce costimulatory or coinhibitory signals. The MIH37 antibody reportedly blocks the binding of CD273 (PB-L2) to its receptors, CD279 (PD-1) or RGMb and can inhibit antigen-specific T cell responses and hapten-induced contact hypersensitivity reactions. The TY25 monoclonal antibody reportedly binds with lower affinity near or at the same mouse CD273 (PD-L2) epitope recognized by the MIH37 antibody.

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 Blue™ 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.

567374 Rev. 1
Format Details
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BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
BV421
Violet 405 nm
407 nm
423 nm
567374 Rev.1
Citations & References
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Development References (6)

  1. Loke P, Allison JP. PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells.. Proc Natl Acad Sci U S A. 2003; 100(9):5336-41. (Biology). View Reference
  2. Matsumoto K, Fukuyama S, Eguchi-Tsuda M, et al. B7-DC induced by IL-13 works as a feedback regulator in the effector phase of allergic asthma.. Biochem Biophys Res Commun. 2008; 365(1):170-5. (Clone-specific: In vivo exacerbation). View Reference
  3. Okudaira K, Hokari R, Tsuzuki Y, et al. Blockade of B7-H1 or B7-DC induces an anti-tumor effect in a mouse pancreatic cancer model. Int J Oncol. 2009; 35(4):741-749. (Clone-specific: Functional assay, In vivo exacerbation). View Reference
  4. Ritprajak P, Hashiguchi M, Akiba H, Yagita H, Okumura K, Azuma M. Antibodies against B7-DC with differential binding properties exert opposite effects. Hybridoma. 2012; 31(1):40-47. (Immunogen: Flow cytometry, Functional assay, Inhibition, In vivo exacerbation). View Reference
  5. Xiao Y, Yu S, Zhu B, et al. RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance. J Exp Med. 2014; 211(5):943-959. (Clone-specific: Blocking). View Reference
  6. Yamazaki T, Akiba H, Iwai H, et al. Expression of programmed death 1 ligands by murine T cells and APC. J Immunol. 2002; 169(10):5538-5545. (Biology). View Reference
View All (6) View Less
567374 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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.