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Flow cytometric analysis of CD279 (PD-1) expression on resting and activated mouse splenocytes. Splenic leucocytes from a C57BL/6 mouse were cultured without (Left Plot) or with (Right Plot) plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 553057) for 3 days. The resting (Left Plot) or activated (Right Plot) splenocytes were harvested, preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142), and stained with either BD Horizon™ BB515 Rat IgG2b, κ Isotype Control (Cat. No. 564421; dashed line histograms) or BD Horizon BB515 Rat Anti-Mouse CD279 (PD-1) antibody (Cat. No. 566832; solid line histograms) at 1 μg/test. BD Via-Probe™ Cell Viability 7-AAD Solution (Cat. No. 555815/555816) was added to cells right before analysis. The fluorescence histograms showing CD279 (PD-1) expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of viable (7-AAD-negative) 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.
BD Horizon™ BB515 Rat Anti-Mouse CD279 (PD-1)
Regulatory Status Legend
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® 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).
For optimal results, it is recommended to perform 2 washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescence staining, prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.
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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- Alexa Fluor™ is a trademark of Life Technologies Corporation.
Companion Products
The RMP1-30 monoclonal antibody specifically recognizes CD279 which is also known as PD-1 (programmed death-1). CD279 (PD-1) is a ~55 kDa type I transmembrane glycoprotein that is encoded by Pdcd1 which belongs to the CD28/CTLA-4 family within the Ig superfamily. CD279 (PD-1) is comprised of an extracellular region with an IgV-like domain and an intracellular region with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) that are associated with inhibitory signaling functions. CD279 (PD-1) is transiently expressed on CD4-CD8- thymocytes and developing B lymphocytes at the pro-B-cell stage. It is also expressed on activated myeloid cells, B cells, and T cells including exhausted T cells found in mice during chronic viral infections or cancer. This co-inhibitory receptor reportedly functions in negative regulation of immune responses and thus helps guard against autoimmunity and preserves peripheral tolerance. 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 that serve as ligands for CD279 (PD-1). The RMP1-30 antibody reportedly does not block the binding of CD279 (PD-1) to these ligands.
Development References (7)
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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. (Biology). View Reference
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Gupta PK, Godec J, Wolski D, et al. CD39 Expression Identifies Terminally Exhausted CD8+ T Cells. PLoS Pathogens. 2015; 11(10):e1005177. (Clone-specific: Flow cytometry). View Reference
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Kasagi S, Kawano S, Okazaki T, et al. Anti-programmed cell death 1 antibody reduces CD4+PD-1+ T cells and relieves the lupus-like nephritis of NZB/W F1 mice. J Immunol. 2010; 184(5):2337-2347. (Clone-specific: Flow cytometry). View Reference
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Liu X, Gibbons RM, Harrington SM, et al. Endogenous tumor-reactive CD8(+) T cells are differentiated effector cells expressing high levels of CD11a and PD-1 but are unable to control tumor growth. Oncoimmunology. 2013; 2(6):e23972. (Clone-specific: Flow cytometry). View Reference
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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. (Immunogen: Flow cytometry, Functional assay). View Reference
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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. (Biology). View Reference
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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. (Biology). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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