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Purified NA/LE Rat Anti-Mouse CD279 (PD-1)

BD Pharmingen™ Purified NA/LE Rat Anti-Mouse CD279 (PD-1)

Clone RMP1-14.rMAb (also known as RMP1-14)

(RUO)
Purified NA/LE Rat Anti-Mouse CD279 (PD-1)
Flow cytometric analysis of PD-1 Ligand (PD-L2 Fc) binding blockade by Purified NA/LE Rat Anti-Mouse CD279 (PD-1) antibody on activated mouse leucocytes. Splenic leucocytes from C57BL/6 mice were cultured with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 553057) for 3 days. The activated leucocytes were harvested and stained with Recombinant Mouse PD-L2/B7-DC (Human IgG1) Fc Chimera Protein (R&D Systems, Cat. No. 1022-PL-100, blue line histogram) to assess PD-1 Ligand binding levels. Activated leucocytes preincubated with either Purified Rat IgG2a, κ Isotype Control (Cat. No. 553927; black line histogram) at 20 µg/test or Purified NA/LE Rat Anti-Mouse CD279 (PD-1) antibody (Cat. No. 566906; red line histogram) at 20 µg/test were also assessed prior to staining with the PD-L2 Fc protein. The cells were washed and stained with R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson Immunoresearch, Cat. No. 109-115-098). The fluorescence histograms showing the bound levels of PD-L2 Fc protein were derived from gated events with the forward and side light-scatter characteristics of viable activated leucocytes. 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 PD-1 Ligand (PD-L2 Fc) binding blockade by Purified NA/LE Rat Anti-Mouse CD279 (PD-1) antibody on activated mouse leucocytes. Splenic leucocytes from C57BL/6 mice were cultured with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 553057) for 3 days. The activated leucocytes were harvested and stained with Recombinant Mouse PD-L2/B7-DC (Human IgG1) Fc Chimera Protein (R&D Systems, Cat. No. 1022-PL-100, blue line histogram) to assess PD-1 Ligand binding levels. Activated leucocytes preincubated with either Purified Rat IgG2a, κ Isotype Control (Cat. No. 553927; black line histogram) at 20 µg/test or Purified NA/LE Rat Anti-Mouse CD279 (PD-1) antibody (Cat. No. 566906; red line histogram) at 20 µg/test were also assessed prior to staining with the PD-L2 Fc protein. The cells were washed and stained with R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson Immunoresearch, Cat. No. 109-115-098). The fluorescence histograms showing the bound levels of PD-L2 Fc protein were derived from gated events with the forward and side light-scatter characteristics of viable activated leucocytes. 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™
PD-1; Pdc1; Pdcd1; mPD-1
Mouse (QC Testing)
Rat SD, also known as Sprague-Dawley (outbred) IgG2a, κ
Mouse PD-1 Transfected Cell Line
Flow cytometry (Routinely Tested), Blocking (Tested During Development)
1.0 mg/ml
No azide/low endotoxin: Aqueous buffered solution containing no preservative, 0.2µm sterile filtered. Endotoxin level is ≤0.1 EU/µg (≤0.01 ng/µg) of protein as determined by the LAL assay.
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.

Recommended Assay Procedures

Clone RMP1-14.rMAb was developed for blocking application. For flow cytometric analysis of mouse CD279 (PD-1), we recommend clones J43 or RMP1-30. Note that clone RMP1-14.rMAb does not block clones J43 or RMP1-30.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
566906 Rev. 3
Antibody Details
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RMP1-14.rMAb

RMP1-14.rMAb is a recombinant monoclonal antibody that specifically recognizes CD279 which is also known as Programmed Death-1 (PD-1). CD279 (PD-1) is a ~55-kDa type I transmembrane glycoprotein that is encoded by Pdcd1 (Programmed cell death 1) which belongs to the CD28/CTLA-4 family of immunoreceptors within the Ig superfamily. CD279 (PD-1) is comprised of an extracellular region with an IgV-like domain, a transmembrane sequence, and an intracellular region with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) that are associated with suppressive immunoregulatory functions. CD279 (PD-1) is variably expressed on some thymocyte subsets and developing B lymphocytes at the pro-B-cell stage. It is also inducibly expressed on activated myeloid cells, B cells, and T cells including exhausted T cells found in mice during chronic viral infections or cancer. Although this co-inhibitory receptor plays roles in mediating immunological tolerance and preventing autoimmune responses it can also inhibit protective immune responses against microbial infections and cancer. CD273 (also known as PD-L2 or B7-DC) and CD274 (PD-L1 or B7-H1) are members of the B7 family within the Ig superfamily. These molecules serve as ligands for CD279 (PD-1) and are variably expressed on lymphoid and nonlymphoid cells types including antigen-presenting cells and tumor cells. The RMP1-14 antibody reportedly blocks the binding of these natural ligands as well as recombinant mouse PD-L1-Ig and PD-L2-Ig proteins to CD279 (PD-1). Antibody-mediated inhibition of the interaction between PD-1 and its ligands can serve as an immune checkpoint blockade that can augment T-cell responses against tumor cells.

                

566906 Rev. 3
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
566906 Rev.3
Citations & References
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Development References (6)

  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. Kondo Y, Ohno T, Nishii N, Harada K, Yagita H, Azuma M. Differential contribution of three immune checkpoint (VISTA, CTLA-4, PD-1) pathways to antitumor responses against squamous cell carcinoma.. Oral Oncol. 2016; 57:54-60. (Clone-specific: Blocking, In vivo exacerbation). View Reference
  3. Matsumoto K, Inoue H, Nakano T, et al. B7-DC regulates asthmatic response by an IFN-gamma-dependent mechanism. J Immunol. 2004; 172(4):2530-2541. (Clone-specific: Blocking, In vivo exacerbation). View Reference
  4. McGee HS, Yagita H, Shao Z, Agrawal DK. Programmed Death-1 antibody blocks therapeutic effects of T-regulatory cells in cockroach antigen-induced allergic asthma.. Am J Respir Cell Mol Biol. 2010; 43(4):432-42. (Clone-specific: Blocking, In vivo exacerbation). View Reference
  5. Seko Y, Yagita H, Okumura K, Azuma M, Nagai R. Roles of programmed death-1 (PD-1)/PD-1 ligands pathway in the development of murine acute myocarditis caused by coxsackievirus B3. Cardiovasc Res. 2007; 75(1):158-167. (Immunogen: Blocking, In vivo exacerbation). View Reference
  6. Totsuka T, Kanai T, Makita S, et al. Regulation of murine chronic colitis by CD4+CD25- programmed death-1+ T cells. Eur J Immunol. 2005; 35(6):1773-1785. (Clone-specific: Blocking, In vivo exacerbation). View Reference
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566906 Rev. 3

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