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PE-CF594 Rat Anti-Mouse CD274
PE-CF594 Rat Anti-Mouse CD274

Two-color flow cytometric analysis of CD274 expression on resting and activated mouse splenocytes. Mouse splenic leucocytes were either not activated (Upper Panels) or activated (Lower Panels) by culture with plate-bound Purified NA/LE Hamster Anti-Mouse CD3 antibody (Cat. No. 553057) for 3 days (37°C). The cells were harvested, washed and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with BD Horizon™ BV421 Rat Anti-Mouse CD4 antibody (Cat. No. 562891) and either BD Horizon™ PE-CF594 Rat IgG2a, κ Isotype Control (Cat. No. 562302; Left Plots) or BD Horizon PE-CF594 Rat Anti-Mouse CD274 antibody (Cat. No. 567032; Right Plots) at 0.5 µg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. Bivariate pseudocolor density plots showing the correlated expression of CD274 (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light- scatter characteristics of viable (7-AAD-negative) 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.

Two-color flow cytometric analysis of CD274 expression on resting and activated mouse splenocytes. Mouse splenic leucocytes were either not activated (Upper Panels) or activated (Lower Panels) by culture with plate-bound Purified NA/LE Hamster Anti-Mouse CD3 antibody (Cat. No. 553057) for 3 days (37°C). The cells were harvested, washed and preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with BD Horizon™ BV421 Rat Anti-Mouse CD4 antibody (Cat. No. 562891) and either BD Horizon™ PE-CF594 Rat IgG2a, κ Isotype Control (Cat. No. 562302; Left Plots) or BD Horizon PE-CF594 Rat Anti-Mouse CD274 antibody (Cat. No. 567032; Right Plots) at 0.5 µg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. Bivariate pseudocolor density plots showing the correlated expression of CD274 (or Ig Isotype control staining) versus CD4 were derived from gated events with the forward and side light- scatter characteristics of viable (7-AAD-negative) 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™
B7-H1, PD-L1; PD1L1; Programmed death ligand 1
Mouse (QC Testing)
Rat SD, also known as Sprague-Dawley (outbred) IgG2a, λ
DBA/2 mouse lymphoma L5178Y transfected with Pdcd1lg1 cDNA
Flow cytometry (Routinely Tested)
0.2 mg/ml
60533
AB_2870014
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 with BD Horizon™ PE-CF594 under optimum conditions, and unconjugated antibody and free PE-CF594 were 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. 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 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.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. Texas Red is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  8. CF™ is a trademark of Biotium, Inc.
  9. When excited by the yellow-green (561-nm) laser, the fluorescence may be brighter than when excited by the blue (488-nm) laser.
  10. This product is provided under an Agreement between BIOTIUM and BD Biosciences. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications owned or licensed by Biotium, Inc. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
  11. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using multi-laser cytometers, which may directly excite both PE and CF™594.
  12. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  13. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
567032 Rev. 1
Antibody Details
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MIH5

The MIH5 monoclonal antibody specifically binds to CD274, also known as B7-H1 or PDL1, a 43-kDa glycoprotein encoded by the Pdcd1lg1 gene of the B7 family of the Ig superfamily. Pdcd1lg1 mRNA is expressed in more tissues than other members of the B7 family; transcripts are found in lymphoid tissues and many, but not all, non-lymphoid tissues. The protein has been detected at low levels on resting peripheral T and B lymphocytes, macrophages, and dendritic cells. B7-H1 mRNA and protein expression are upregulated upon activation of T and B cells, macrophages, dendritic cells, and epidermal keratinocytes by a variety of stimulatory factors. B7-H1's receptor, PD-1, contains an ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) on its intracytoplasmic region and is expressed on activated B and T lymphocytes, suggesting that B7-H1-PD-1 interaction may be involved in the negative regulation of immune responses. The second PD-1 ligand, B7-DC (PD-L2), is also a member of the B7 family of the Ig superfamily. Furthermore, B7-H1 may participate in positive immunoregulation, or costimulation of T cells, through an additional receptor, which is not PD-1 and distinct from the alternate receptor for B7-DC. The MIH5 antibody blocks the binding of PD-1-Ig to B7-H1 transfectants.

This antibody is conjugated to BD Horizon PE-CF594, which has been developed exclusively by BD Biosciences as a better alternative to PE-Texas Red®. PE-CF594 excites and emits at similar wavelengths to PE-Texas Red® yet exhibits improved brightness and spectral characteristics. Due to PE having maximal absorption peaks at 496 nm and 564 nm, PE-CF594 can be excited by the blue (488-nm), green (532-nm) and yellow-green (561-nm) lasers and can be detected with the same filter set as PE-Texas Red® (eg, 610/20-nm filter).

567032 Rev. 1
Format Details
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PE-CF594
BD Horizon™ PE-CF594 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye with an emission maximum (Em Max) at 615-nm. PE-CF594, driven by BD innovation, is designed to be excited by the blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 615 nm (e.g., a 610/20-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the green (532-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE-CF594
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
615 nm
567032 Rev.1
Citations & References
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Development References (9)

  1. Ansari MJ, Salama AD, Chitnis T, et al. The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med. 2003 July; 198(1):63-69. (Biology). View Reference
  2. Carreno BM, Collins M. The B7 family of ligands and its receptors: New pathways for costimulation and inhibition of immune responses. Annu Rev Immunol. 2002; 20:29-53. (Biology). View Reference
  3. Dong H, Chen L. B7-H1 pathway and its role in the evasion of tumor immunity. J Mol Med. 2003 May; 81(5):281-287. (Biology). View Reference
  4. Hessel EM, Chu M, Lizcano JO, et al. Immunostimulatory oligonucleotides block allergic airway inflammation by inhibiting Th2 cell activation and IgE-mediated cytokine induction. J Exp Med. 2005; 202(11):1563-1573. (Clone-specific: Flow cytometry). View Reference
  5. Liu X, Gao JX, Wen J, et al. B7DC/PDL2 promotes tumor immunity by a PD-1-independent mechanism. J Exp Med. 2003; 197:1721-1730. (Biology). View Reference
  6. Tamura H, Dong H, Zhu G, et al. B7-H1 costimulation preferentially enhances CD28-independent T-helper cell function. Blood. 2001; 97(6):1809-1816. (Biology). View Reference
  7. Tsushima F, Iwai H, Otsuki N, et al. Preferential contribution of B7-H1 to programmed death-1-mediated regulation of hapten-specific allergic inflammatory responses. Eur J Immunol. 2003; 33(10):2773-2782. (Immunogen: Blocking, Enhancement, Functional assay, Immunofluorescence, Immunohistochemistry, Inhibition, In vivo exacerbation). View Reference
  8. Wang S, Bajorath J, Flies DB, Dong H, Honjo T, Chen L. Molecular modeling and functional mapping of B7-H1 and B7-DC uncouple costimulatory function from PD-1 interaction. J Exp Med. 2003; 197:1083-1091. (Biology). View Reference
  9. 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 (9) View Less
567032 Rev. 1

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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.