Cd274 pd l1 apc

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CD274/PD-L1/APC is a fluorochrome-conjugated antibody that binds to the CD274 (also known as PD-L1) protein. CD274 is a cell surface glycoprotein that plays a role in the regulation of the immune response.

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Close spelling variants of this product

PD-L1 (CD274) (3 citations) PD-L1-APC (17 citations) CD274-APC (2 citations) PD-L1 (75 citations) CD274 (9 citations)

6 protocols using cd274 pd l1 apc

Multiparametric Flow Cytometry Analysis

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Flow cytometry was performed using the following antibodies: HLA-DR/PE-Cy7 (Biolegend, clone L243, 1:20), CD274/PD-L1/APC (Biolegend, clone 29E.2A3, 1:200) and HLA-A/B/C –Alexa Fluor488 (1:100, Biolegend, clone W6/32) mouse MHC-II (I-A/I-E, 1:20, Biolegend, clone M5/114.15.2). DAPI was used as a viability dye. Samples were analysed on an Aria III laser system (BD Biosciences).

Johnson D.B., Estrada M.V., Salgado R., Sanchez V., Doxie D.B., Opalenik S.R., Vilgelm A.E., Feld E., Johnson A.S., Greenplate A.R., Sanders M.E., Lovly C.M., Frederick D.T., Kelley M.C., Richmond A., Irish J.M., Shyr Y., Sullivan R.J., Puzanov I., Sosman J.A, & Balko J.M. (2016). Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy. Nature Communications, 7, 10582.

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Analyzing PD-1 and PD-L1 Expression in RA

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PD-1 expression on CD4 T cells and PD-L1 expression on CD1c mDCs of RA patients were analysed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Ex vivo or cultured mDCs were stained with CD1c phycoerythrin (CD1c-PE; BD Biosciences), CD19 peridinin chlorophyll (CD19-PerCP; BioLegend) and CD274 (PD-L1)-APC (BioLegend). mDCs were gated as CD1c-positive and CD19-negative. Ex vivo CD4 T cells were stained with CD45RO fluorescein isothiocyanate (CD45RO-FITC; Dako, Glostrup, Denmark), CD27-APC (Invitrogen), CD279 (PD-1)-PE and CD4-PerCP (BioLegend) using isotype antibodies or autofluorescence as controls. All samples were analysed using FlowJo software (TreeStar, Ashland, OR, USA). To compare mean fluorescence intensity (MFI) values, the autofluorescence intensity was subtracted from the MFI of the stains to reveal true expression values.

Moret F.M., van der Wurff-Jacobs K.M., Bijlsma J.W., Lafeber F.P, & van Roon J.A. (2014). Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/PD-L1 interactions. Arthritis Research & Therapy, 16(6), 497.

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Multiparameter Flow Cytometry for Immune Profiling

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The following monoclonal antibodies and reagents were used with the appropriate isotype controls and were obtained from BD Biosciences unless otherwise stated. Human immunoglobulin κ light chain allophycocyanin (APC) (catalog no. 561323), CD45 AlexaFluor 700 (catalog no. 56056), CD19 APC-Cy7 (Biolegend, catalog no. 348794), human immunoglobulin λ light chain AlexaFluor 488 (Biolegend, catalog no. 316612), CD138 PerCP-Cy5.5 (catalog no. 564605), CD38 BV421 (catalog no. 562444), LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, catalog no. L34957), CD14 V500 (catalog no. 561391), CD56 BV605 (catalog no. 562780), CD20 BV650 (catalog no. 563780), CD3 BV711 (Biolegend, catalog no. 317328), BCMA PE (Biolegend, catalog no. 357504), CD200 PE-Cy7 (Biolegend, catalog no. 329212), CD200 PE (Biolegend, catalog no. 329306), CD200R PE-Cy7 (Biolegend, catalog no. 329312), CD200R APC (Biolegend, catalog no. 329307), CD200R PE (Biolegend), CD3 BV421 (catalog no. 562427), CD4 BV785 (Biolegend, catalog no. 317442), CD8 APC-Cy7 (catalog no. 557834), CD197(CCR7) FITC (catalog no. 561271), CD45RO PE-Cy7(Biolegend, catalog no. 304230), and CD274(PD-L1) APC (catalog no. 563741). The data were acquired with BD Fortessa and analyzed with FlowJo (version 10).

Tang Y., Liu W., Kadu S., Johnson O., Hasanali Z.S., Kelly A., Shestov A., Pajarillo R., Greenblatt E., Holmes M., Wang L.P., Shih N., O’Connor R.S., Ruella M., Garfall A.L., Allman D., Vogl D.T., Cohen A., June C.H, & Sheppard N.C. (2023). Exploiting the CD200-CD200R immune checkpoint axis in multiple myeloma to enhance CAR T-cell therapy. Blood, 143(2), 139-151.

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Multiparameter Flow Cytometry of Immune Cells

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Cells were washed in phosphate-buffered saline (PBS) and harvested with Accutase (MilliporeSigma, SCR005) for 10 minutes at room temperature. Dissociated cells were washed once in flow staining buffer (PBS + 1% FBS) and incubated with respective flow antibodies at 4°C for 20 minutes in the dark. Flow cytometry was performed using the following antibodies: CD45/AF488 (BioLegend clone 30-F11, 1:500), Ly6C/PE (BioLegend clone HK1.4, 1:250), Ly6G/APC-Cy7 (BioLegend clone 1A8,1:250), CD11b/PE-Cy7 (BioLegend clone M1/70,1:250), CD274 (PD-L1)/APC (BioLegend clone 10F.9G2, 1:250), and mouse MHC-II (I-A/I-E)/BV711 (BioLegend clone M5/114.15.2, 1:500). DAPI was used as a viability dye for dead cell exclusion. Samples were analyzed on an Attune NxT flow cytometer (Life Technologies, Thermo Fisher Scientific).

Franklin D.A., Sharick J.T., Ericsson-Gonzalez P.I., Sanchez V., Dean P.T., Opalenik S.R., Cairo S., Judde J.G., Lewis M.T., Chang J.C., Sanders M.E., Cook R.S., Skala M.C., Bordeaux J., Orozco Bender J., Vaupel C., Geiss G., Hinerfeld D, & Balko J.M. (2020). MEK activation modulates glycolysis and supports suppressive myeloid cells in TNBC. JCI Insight, 5(15), e134290.

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Multiparametric flow cytometry for immune cell profiling

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Flow cytometry was used to determine protein levels on living cells. The following extra-cellular antibodies were used: CD3-APC (clone: 17A2), CD8-FITC (clone: 53-6.7), Rat IgG1, κ-PE isotype control (clone: eBRG1), from eBioscience; CD8a-APC (clone: 53-6.7), PD-1 (CD279)-PE_Cy7 (clone: 29F.1a12), PD-L1 (CD274)-APC (clone: 10F.9G2), CD4-Alexafluor647 (clone: GK1.5), Rat IgG2a, κ-FITC (clone: RTK2758), Rat IgG2b-Alexafluor 647(clone: RTK4530), Rat IgG2b, κ-APC (clone: RTK4530), κ-FITC (clone:RTK2758), from Biolegend. For intracellular protein staining we used anti-IFN-γ-Alexafluor 647 (clone: XMG1.2), anti-IFN-γ-PE (clone: XMG1.2) from eBioscience. Cell viability was monitored with either propidium iodide, from Sigma-Aldrich, Fixable Viability Dye -eFluor780, from eBioscience, or Aquablue Live/Dead Stain from ThermoFisher Scientific; depending on the experiment requirements. Data were acquired on a Fortessa (BD Biosciences) or a CyAn ADP (Beckman Coulter) and analyzed using FlowJo v10.

Mastroianni J., Stickel N., Andrlova H., Hanke K., Melchinger W., Duquesne S., Schmidt D., Falk M., Andrieux G., Pfeifer D., Dierbach H., Schmitt-Graeff A., Meiss F., Boerries M, & Zeiser R. (2018). miR-146a controls immune response in the melanoma microenvironment. Cancer research, 79(1), 183-195.

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Characterization of CAR T-cells and PD1-VHH

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The antibodies against the following markers were used in this study (all anti-human): CD3 APC/Cy7 (Biolegend), CD19 PE (Biolegend), PD-1 (CD279) PE (BD Biosciences) and FITC (Biolegend), PD-L1 (CD274) APC (Biolegend), CD45RA PE (Biolegend), CD62L APC (Biolegend), TIGIT PE/Cy7 (Biolegend). The CAR molecules were detected by staining with CD19 CAR Detection Reagent (Biotin) (Miltenyi Biotec), or FITC-Labeled Human CD19 (Acro Biosystems), for primary staining and streptavidin-APC/Cy7 or FITC (Abcam) for visualization. The PD1-VHH-Fc-PDGFR molecules were detected using goat cross-absorbed anti-human IgG antibody conjugated with DyLight650 (Thermo Fisher Scientific). Flow cytometric analysis was performed using NovoCyte 2060 (ACEA Biosciences) instrument, data were analyzed with FlowJo X10 (FlowJo) and NovoExpress Software (ACEA Biosciences). Jurkat-Lucia™ NFAT and Nalm-6 were transduced with the pLV₂ PD-1 and pLV₂ PD-L1 lentiviruses, respectively, and positive cells were sorted using SH800 (Sony) instrument.

Kalinin R.S., Ukrainskaya V.M., Chumakov S.P., Moysenovich A.M., Tereshchuk V.M., Volkov D.V., Pershin D.S., Maksimov E.G., Zhang H., Maschan M.A., Rubtsov Y.P, & Stepanov A.V. (2021). Engineered Removal of PD-1 From the Surface of CD19 CAR-T Cells Results in Increased Activation and Diminished Survival. Frontiers in Molecular Biosciences, 8, 745286.

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