BD Horizon™ RB780 Rat Anti-Mouse CD274 (PD-L1)
Clone 10F.9G2 (RUO)
Multicolor flow cytometric analysis of CD274 (PD-L1) expression on resting and activated viable Mouse splenic leucocytes. BALB/c Mouse splenocytes were either not activated (Upper Plots) or activated (Lower Plots) by culture with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 567114/567115/553057) 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 APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051/561091) and with either BD Horizon™ RB780 Rat IgG2b, κ Isotype Control (Cat. No. 568699; Left Plots) or BD Horizon™ RB780 Rat Anti-Mouse CD274 (PD-L1) antibody (Cat. No. 569146/569147; 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. The bivariate pseudocolor density plots showing the correlated expression of CD274 (PD-L1) [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™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
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Multicolor flow cytometric analysis of CD274 (PD-L1) expression on resting and activated viable Mouse splenic leucocytes. BALB/c Mouse splenocytes were either not activated (Upper Plots) or activated (Lower Plots) by culture with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e antibody (Cat. No. 567114/567115/553057) 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 APC Rat Anti-Mouse CD4 antibody (Cat. No. 553051/561091) and with either BD Horizon™ RB780 Rat IgG2b, κ Isotype Control (Cat. No. 568699; Left Plots) or BD Horizon™ RB780 Rat Anti-Mouse CD274 (PD-L1) antibody (Cat. No. 569146/569147; 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. The bivariate pseudocolor density plots showing the correlated expression of CD274 (PD-L1) [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™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
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 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.
Product Notices
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- 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.
- 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.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
Companion Products
The 10F.9G2 monoclonal antibody specifically recognizes Programmed cell death 1 ligand 1 (PDCD1 ligand 1) which is also known as Programmed death ligand 1 (PD-L1), B7 homolog 1 (B7-H1), or CD274. CD274 (PD-L1) is a 43-kDa type I transmembrane glycoprotein that is encoded by Cd274 which belongs to the B7 family within the Ig superfamily. CD274 (PD-L1) is expressed at low levels on resting peripheral T and B lymphocytes, monocytes, macrophages, dendritic cells (DC), and NK cells and undergoes upregulated expression upon cellular activation. PD-L1 (CD274) is also widely expressed on nonhematopoietic cell types including epithelial cells, endothelial cells, placental trophoblasts, and tumor cells. CD274 (PD-L1) and CD273 (PD-L2) serve as ligands for the CD279 (Programmed cell death protein 1/PD-1) inhibitory receptor. CD274 (PD-L1)-mediated signaling through CD279 (PD-1) regulates T cell responses in lymphoid and nonlymphoid tissues that are important for ensuring protective immunity while maintaining peripheral tolerance. This immune signaling checkpoint may suppress antitumor immune responses and prevent tumor rejection. The 10F.9G2 antibody reportedly blocks CD274 (PD-L1) binding to CD279 (PD-1) and can enhance the proliferation and effector responses of activated T cells including cytokine production and cytolytic activity.
Development References (6)
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Eppihimer MJ, Gunn J, Freeman GJ, et al. Expression and regulation of the PD-L1 immunoinhibitory molecule on microvascular endothelial cells. Microcirculation. 2002; 9(2):133-145. (Immunogen: ELISA, Flow cytometry, Immunohistochemistry, Radioimmunoassay). View Reference
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Freeman GJ, Long AJ, Iwai Y, et al. Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000; 192:1027-1034. (Biology). View Reference
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Iraolagoitia XL, Spallanzani RG, Torres NI, et al. NK Cells Restrain Spontaneous Antitumor CD8+ T Cell Priming through PD-1/PD-L1 Interactions with Dendritic Cells.. J Immunol. 2016; 197(3):953-61. (Clone-specific: Flow cytometry). View Reference
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Paterson AM, Brown KE, Keir ME, et al. The programmed death-1 ligand 1:B7-1 pathway restrains diabetogenic effector T cells in vivo.. J Immunol. 2011; 187(3):1097-105. (Clone-specific: Blocking). View Reference
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Rodig N, Ryan T, Allen JA, et al. Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis.. Eur J Immunol. 2003; 33(11):3117-26. (Clone-specific: Blocking, Flow cytometry, Functional assay). View Reference
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Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection.. Nat Immunol. 2007; 8(3):239-45. (Biology). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.