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Purifed NA/LE Hamster Anti-Mouse CD279 (PD-1)
Purifed NA/LE Hamster Anti-Mouse CD279 (PD-1)

Flow cytometric analysis of CD279 (PD-1) expression on activated mouse splenocytes. C57BL/6 mouse splenic leucocytes were cultured with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 553057) for 3 days. Activated splenocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with either Purified Hamster IgG2, κ Isotype Control (Cat. No. 559277; dashed line histogram) or Purified NA/LE Hamster Anti-Mouse CD279 (PD-1) antibody (Cat. No. 566823; solid line histogram) at 1 µg/test. The cells were washed and stained with Biotin Mouse Anti-Hamster IgG (Cat. No. 54025) followed by PE Streptavidin (Cat. No. 554061). The fluorescence histograms showing CD276 (PD-1) expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of viable splenocytes. 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.

Purifed NA/LE Hamster Anti-Mouse CD279 (PD-1)

Flow cytometric analysis of J43 antibody blocking of ligand binding to CD279 (PD-1) on activated mouse splenocytes. C57BL/6 mouse splenic leucocytes were activated in culture with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 553057) for 3 days.

       To assess ligand binding to cells expressing CD279 (PD-1), the activated splenocytes were stained with either Recombinant Mouse PD-L1/B7-H1 Fc Chimera Protein (PD-L1-Fc; R&D Systems, Cat. No. 1019-B7-100; blue line histogram, Left Plot) or Recombinant Mouse PD-L2/B7-DC Fc Chimera Protein (PD-L2-Fc; R&D Systems, Cat. No. 1022-PL-100; blue line histogram, Right Plot). This was followed by secondary staining of the bound recombinant ligands with PE AffiniPure Goat Anti-Human IgG, Fc Gamma Fragment Specific antiserum (PE Anti-Human IgG, Fc; Jackson Immunoresearch, Cat. No. 109-115-098).

       To assess the PD-1 ligand-blocking activity of clone J43, the activated cells were preincubated with either Purified Hamster IgG2, κ Isotype Control (Cat. No. 559277; black line histograms at 20 µg/test) or Purified NA/LE Hamster Anti-Mouse CD279 (PD-1) antibody (Cat. No. 566823; red line histograms at 20 µg/test) prior to staining with PD-L1 Fc (Left Plots) and PD-L2 Fc (Right Plots). The cells were then washed and secondarily stained with PE Anti-Human IgG, Fc.

       The fluorescence histograms showing either PD-L1-Fc (Left Plots) or PD-L2-Fc (Right Plots) staining of cells were derived from gated events with the forward and side light-scatter characteristics of viable splenocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 and FlowJo® software. Data shown on this Technical Data Sheet are not lot specific.

Flow cytometric analysis of CD279 (PD-1) expression on activated mouse splenocytes. C57BL/6 mouse splenic leucocytes were cultured with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 553057) for 3 days. Activated splenocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with either Purified Hamster IgG2, κ Isotype Control (Cat. No. 559277; dashed line histogram) or Purified NA/LE Hamster Anti-Mouse CD279 (PD-1) antibody (Cat. No. 566823; solid line histogram) at 1 µg/test. The cells were washed and stained with Biotin Mouse Anti-Hamster IgG (Cat. No. 54025) followed by PE Streptavidin (Cat. No. 554061). The fluorescence histograms showing CD276 (PD-1) expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of viable splenocytes. 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.

Flow cytometric analysis of J43 antibody blocking of ligand binding to CD279 (PD-1) on activated mouse splenocytes. C57BL/6 mouse splenic leucocytes were activated in culture with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 553057) for 3 days.

       To assess ligand binding to cells expressing CD279 (PD-1), the activated splenocytes were stained with either Recombinant Mouse PD-L1/B7-H1 Fc Chimera Protein (PD-L1-Fc; R&D Systems, Cat. No. 1019-B7-100; blue line histogram, Left Plot) or Recombinant Mouse PD-L2/B7-DC Fc Chimera Protein (PD-L2-Fc; R&D Systems, Cat. No. 1022-PL-100; blue line histogram, Right Plot). This was followed by secondary staining of the bound recombinant ligands with PE AffiniPure Goat Anti-Human IgG, Fc Gamma Fragment Specific antiserum (PE Anti-Human IgG, Fc; Jackson Immunoresearch, Cat. No. 109-115-098).

       To assess the PD-1 ligand-blocking activity of clone J43, the activated cells were preincubated with either Purified Hamster IgG2, κ Isotype Control (Cat. No. 559277; black line histograms at 20 µg/test) or Purified NA/LE Hamster Anti-Mouse CD279 (PD-1) antibody (Cat. No. 566823; red line histograms at 20 µg/test) prior to staining with PD-L1 Fc (Left Plots) and PD-L2 Fc (Right Plots). The cells were then washed and secondarily stained with PE Anti-Human IgG, Fc.

       The fluorescence histograms showing either PD-L1-Fc (Left Plots) or PD-L2-Fc (Right Plots) staining of cells were derived from gated events with the forward and side light-scatter characteristics of viable splenocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 and FlowJo® software. Data shown on this Technical Data Sheet are not lot specific.

Product Details
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BD Pharmingen™
Ly101; PD-1; Pdc1; Pdcd1; mPD-1; programmed cell death 1 protein; programmed cell death protein 1; protein PD-1
Mouse (QC Testing)
Armenian Hamster IgG2, κ
Syrian Hamster kidney cell line BKH transfected with Pdcd1 cDNA
Flow cytometry (Routinely Tested), Blocking (Tested During Development)
1.0 mg/ml
18566
AB_2869886
Aqueous buffered solution containing no preservative.
RUO


Preparation And Storage

Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. This preparation contains no preservatives, thus it should be handled under aseptic conditions.

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. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  4. Although hamster immunoglobulin isotypes have not been well defined, BD Biosciences Pharmingen has grouped Armenian and Syrian hamster IgG monoclonal antibodies according to their reactivity with a panel of mouse anti-hamster IgG mAbs. A table of the hamster IgG groups, Reactivity of Mouse Anti-Hamster Ig mAbs, may be viewed at http://www.bdbiosciences.com/documents/hamster_chart_11x17.pdf.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
566823 Rev. 1
Antibody Details
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J43

The J43 monoclonal antibody specifically recognizes CD279 which is also known as PD-1 (programmed death-1). CD279 is a 50-55-kDa glycoprotein encoded by the Pdcd1 gene of the CD28 family of the Ig superfamily. The expression of Pdcd1 mRNA and PD-1 protein is tightly regulated. PD-1 is transiently expressed on CD4-CD8 thymocytes, it is upregulated on some cell lines upon induction of apoptosis, it is induced on thymocytes and splenic T and B lymphocytes after stimulation through their antigen receptors, and it is induced on activated myeloid cells. In addition, Pdcd1 mRNA is transiently expressed in developing B lymphocytes at the pro-B-cell stage. The presence of an ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) on PD-1's intracytoplasmic region and the development of splenomegaly and breakdown of peripheral tolerance in PD-1[-/-] mice suggest that PD-1 is involved in the negative regulation of immune responses. The PD-1 ligands, B7-H1 (also known as PD-L1, CD274) and B7-DC (PD-L2, CD273), are members of the B7 family of the Ig superfamily. The J43 antibody blocks the binding of PD-1 to its two ligands.

        

566823 Rev. 1
Format Details
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NA/LE
NA/LE refers to the culture and purification methods and buffer used to produce purified antibodies with no azide and low endotoxin: Aqueous buffered solution containing no preservative, 0.2µm sterile filtered. Endotoxin level is ≤0.01 EU/µg (≤0.001 ng/µg) of protein as determined by the LAL assay.NA/LE are perfectly suited to be used in culture or in vivo (for nonhuman studies) for functional assays — blocking, neutralizing, activation or depletion — where the presence of azide may damage cells or exogenous endotoxin may signal or activate cells.
NA/LE
566823 Rev.1
Citations & References
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Development References (8)

  1. Agata Y, Kawasaki A, Nishimura H, et al. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol. 1996 May; 8(5):765-772. (Immunogen: Flow cytometry, Immunoprecipitation). View Reference
  2. 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. (Clone-specific: Blocking). View Reference
  3. 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
  4. Finger LR, Pu J, Wasserman R, et al. The human PD-1 gene: complete cDNA, genomic organization, and developmentally regulated expression in B cell progenitors. Gene. 1997 September; 197(1-2):177-187. (Biology). View Reference
  5. Nishimura H, Agata Y, Kawasaki A, et al. Developmentally regulated expression of the PD-1 protein on the surface of double-negative (CD4-CD8-) thymocytes. Int Immunol. 1996 May; 8(5):773-780. (Clone-specific: Flow cytometry). View Reference
  6. Nishimura H, Minato N, Nakano T, Honjo T. Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell responses. Int Immunol. 1998; 10(10):1563-1572. (Clone-specific: Flow cytometry). View Reference
  7. Salama AD, Chitnis T, Imitola J, et al. Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis. J Exp Med. 2003 July; 198(1):71-78. (Clone-specific: Blocking). View Reference
  8. 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. (Clone-specific). View Reference
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566823 Rev. 1

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