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Pd 1 clone eh12.2h7

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PD-1 (clone EH12.2H7) is a monoclonal antibody that specifically binds to the human programmed cell death-1 (PD-1) receptor. PD-1 is an immune checkpoint protein expressed on the surface of activated T cells, B cells, and myeloid cells.

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Lab products found in correlation

Anti cd16 cd32 clone 93, by BioLegend (2 mentions) True nuclear transcription factor buffer, by BioLegend (2 mentions) Zombie nir fixable viability dye, by BioLegend (2 mentions) Lsrfortessa, by BD (2 mentions) Cd4 clone l200, by BD (2 mentions) Hla dr clone l243, by BD (2 mentions) Clone sp34 2, by BD (1 mentions) Cd45ro clone uchl1, by Beckman Coulter (1 mentions) Cd27 clone m t271, by BD (1 mentions) Tq prep machine, by Beckman Coulter (1 mentions) Cd8 clone rpa t8, by BioLegend (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Fix perm cell permeabilisation kit, by Thermo Fisher Scientific (1 mentions) Fortessa, by BD (1 mentions) Brefeldin a, by Merck Group (1 mentions) Facsdiva software, by BD (1 mentions) Tnf α, by BD (1 mentions) Ifn γ, by BD (1 mentions) Foxp3 staining buffer set, by Miltenyi Biotec (1 mentions) Interleukin 2 (il 2), by BD (1 mentions)

Close spelling variants of this product

PD-1 (EH12.2H7, (20 citations) EH12.2H7 (13 citations) Anti-PD-1 (clone EH12.2H7, (9 citations) PD-1-BV421 (clone EH12.2H7) (9 citations) Clone EH12.2H7 (5 citations) PD-1 (EH12.1) (4 citations) Anti-human PD-1 (clone EH12.2H7, (4 citations) Anti-PD-1 BV421 (clone EH12.2H7), (3 citations) Human PD-1 (clone EH12.2H7, (3 citations) PD-1 (FITC; clone: EH12.2H7; (2 citations)

9 protocols using pd 1 clone eh12.2h7

1

Multiparametric Analysis of Activated T Cells

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Following treatment, anti-CD3/CD28 stimulated and unstimulated Jurkat T cells were transferred to 96-well V-bottom plates and washed twice with PBS containing 0.1% BSA and 5 mM EDTA (PBS + BSA + EDTA). Cells were then incubated with anti-CD16/CD32 (clone 93, Biolegend) to prevent nonspecific binding. Single-cell suspensions were stained with antibodies against CD25 (clone BC96), CD38 (clone HB-7), CD69 (clone FN50), CD82 (clone ASL-24), CD154 (clone 24-31), PD-1 (clone EH12.2H7), and Zombie NIR fixable viability dye (all from Biolegend) for 30 min at 4 °C. Cells were washed twice with PBS + BSA + EDTA and fixed in Biolegend fixation/permeabilization buffer for 1 h at room temperature followed by permeabilization using True-Nuclear Transcription Factor buffer (Biolegend) for 45 min in the presence of antibody against Ki-67 (clone Ki-67). Cells were washed twice with permeabilization buffer, acquired with a LSRFortessa (BD) cell analyzer, and data analysis was performed with FlowJo (Tree Star) software.
Datlinger P., Rendeiro A.F., Schmidl C., Krausgruber T., Traxler P., Klughammer J., Schuster L.C., Kuchler A., Alpar D, & Bock C. (2017). Pooled CRISPR screening with single-cell transcriptome read-out. Nature methods, 14(3), 297-301.
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2

Multicolor Flow Cytometry Analysis of T-cell Subsets

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A 100μl aliquot of whole blood was stained with fluorescently-conjugated antibodies specific for human CD3 (clone SP34.2 BD Biosciences), CD4 (clone L200 BD Biosciences), CD8 (clone RPA-T8 BioLegend), CD45RO (clone UCHL1 Beckman Coulter), CD27 (clone M-T271 BD Biosciences), CD38 (clone HB7 BD Biosciences), HLA-DR (clone L243 BD Biosciences), and PD-1 (clone EH12.2H7 BioLegend). Samples were run through TQ-Prep machine (Beckman Coulter) to lyse red blood cells, washed in D-PBS, and fixed in 1.5% formaldehyde. Data were collected by running the samples through multicolor flow cytometry on a LSRII flow cytometer (BD Biosciences), and analyzed with FlowJo software (Treestar Inc.) The gating strategy was as follows:T-cells were identified by forward vs side scatter, excluded of doublets, and further gated on CD3+ cells, which were then subdivided into CD3+/CD4+ and CD3+/CD8+ populations. Those, in turn, were subdivided by receptor status into naïve cells (CD45RO-/CD27+), effector cells (CD45RO-/CD27-), effector memory cells (CD45RO+/CD27-) and central memory cells (CD45RO+/CD27+). Each subset was evaluated for expression of activation markers CD38 and HLA-DR, and for exhaustion marker PD-1.
DeWolfe D., Gandhi J., Mackenzie M.R., Broge TA J.r., Bord E., Babwah A., Mandelbrot D.A., Pavlakis M., Cardarelli F., Viscidi R., Chandraker A, & Tan C.S. (2017). Pre-transplant immune factors may be associated with BK polyomavirus reactivation in kidney transplant recipients. PLoS ONE, 12(5), e0177339.
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3

Multi-parameter Immune Profiling of Skin and Blood

Cited 2 times
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Multi-parameter analysis of skin and blood T cell phenotype was performed on LSR II or BD Fortessa using FACS Diva software (both BD Biosciences, Oxford, UK) and further analyzed using FlowJo software (TreeStar, Inc) as previously described (Agius et al., 2009 (link); Vukmanovic-Stejic et al., 2013 (link)). PBMCs or skin cells were stained with different combinations of antibodies including CD3, CD4, CD8, CD45RA, CD28, CD27 (BD Biosciences) Ki67, CLA, CCR7 and PD-1 (clone EH12.2H7; Biolegend, London, UK). All surface staining was performed for 30 minutes on ice. Isotype control staining and FMO controls were used to set the quadrants. Ki67 staining (clone B56, BD Bioscience) was performed by intracellular staining using the Foxp3 Staining Buffer Set (Miltenyi Biotec, Bisley, UK). For intracellular cytokine staining cells were stimulated with VZV lysate (Virusys corporation, Taneytown, MD) or SEB as positive control and incubated for 15 hours at 37°C, 5% CO2 in the presence of 5 μg/ml Brefeldin A (Sigma-Aldrich, Gillingham, UK). Unstimulated controls were always included. Following stimulations cells were stained for surface markers for 30 minutes at 4°C, washed, fixed and permeabilised (Fix & Perm Cell Permeabilisation Kit, Invitrogen, Paisley, UK) before staining for IL-2, IFN-γ and TNF-α (all from BD Biosciences, Oxford, UK).
Vukmanovic-Stejic M., Sandhu D., Seidel J.A., Patel N., Sobande T.O., Agius E., Jackson S.E., Fuentes-Duculan J., Suarez-Farinas M., Mabbott N.A., Lacy K.E., Ogg G., Nestle F.O., Krueger J.G., Rustin M.H, & Akbar A.N. (2015). The Characterization of Varicella Zoster Virus Specific T Cells In Skin and Blood During Ageing. The Journal of investigative dermatology, 135(7), 1752-1762.
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4

Multiparametric Analysis of Activated T Cells

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Following treatment, anti-CD3/CD28 stimulated and unstimulated Jurkat T cells were transferred to 96-well V-bottom plates and washed twice with PBS containing 0.1% BSA and 5 mM EDTA (PBS + BSA + EDTA). Cells were then incubated with anti-CD16/CD32 (clone 93, Biolegend) to prevent nonspecific binding. Single-cell suspensions were stained with antibodies against CD25 (clone BC96), CD38 (clone HB-7), CD69 (clone FN50), CD82 (clone ASL-24), CD154 (clone 24-31), PD-1 (clone EH12.2H7), and Zombie NIR fixable viability dye (all from Biolegend) for 30 min at 4 °C. Cells were washed twice with PBS + BSA + EDTA and fixed in Biolegend fixation/permeabilization buffer for 1 h at room temperature followed by permeabilization using True-Nuclear Transcription Factor buffer (Biolegend) for 45 min in the presence of antibody against Ki-67 (clone Ki-67). Cells were washed twice with permeabilization buffer, acquired with a LSRFortessa (BD) cell analyzer, and data analysis was performed with FlowJo (Tree Star) software.
Datlinger P., Rendeiro A.F., Schmidl C., Krausgruber T., Traxler P., Klughammer J., Schuster L.C., Kuchler A., Alpar D, & Bock C. (2017). Pooled CRISPR screening with single-cell transcriptome read-out. Nature methods, 14(3), 297-301.
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5

Multiparameter Phenotypic Characterization of PBMCs

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Thawed PBMCs were surface stained with Aqua amine reactive viability dye (Invitrogen) and mAbs to CD45 (clone D058–1283, BD Biosciences), CD3 (clone SP34–2, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone 3B5, Invitrogen), CD95 (clone DX2, BioLegend), CD28 (clone CD28.2, Beckman), CD127 (clone hIL-7R-M21, BD Biosciences), PD-1 (clone EH12.2H7, BioLegend), and HLA-DR (clone L243, BD Biosciences) for 20 min at room temperature, fixed, and then permeabilized with FoxP3 Fix/Perm Buffers (eBioscience) per the manufacturer’s instructions. Permeabilized cells were then stained intracellularly for Ki67 (clone B56, BD Biosciences) and FoxP3 (clone PCH101, eBioscience) for 30 min at 4°C, washed in FoxP3 Perm/Wash buffer, fixed in 0.5% paraformaldehyde and analyzed by flow cytometry on a BD LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo Software (Tree Star Inc).
Swainson L.A., Ahn H., Pajanirassa P., Khetarpal V., Deleage C., Estes J.D., Hunt P.W., Munoz-Sanjuan I, & McCune J.M. (2019). Kynurenine 3-monooxygenase inhibition during acute SIV infection lowers PD-1 expression and improves post-cART CD4+ T cell counts and body weight: KMO inhibition improves clinical outcome in SIV infection. Journal of immunology (Baltimore, Md. : 1950), 203(4), 899-910.
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6

Multiparametric Flow Cytometry Analysis

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Cells were resuspended in FACS staining buffer (PBS + 3% fetal bovine serum) using the following antibodies: CD3 (clone OKT3, BD Biosciences 555342, diluted 1:50), PD1 (clone EH12.2H7, BioLegend, 329928, diluted 1:80), Tim3 (clone 7D3, BD Biosciences 565566, diluted 1:100), CD22 (clone HIB22, BD Biosciences 562860, diluted 1:100), CD19 (clone FMC63, Novus Biologicals 52716, diluted 1:80-1:32000; clone HIB19, BD Biosciences 555413, diluted 1:50), CD107a-PECy7 (clone H4A3, Biolegend 328607, diluted 1:100), CD34 APC (BD 555824, diluted 1:50). CARs transduction was evaluated by staining for a truncated CD34 selection marker located downstream of a P2A ribosomal skip sequence from the CAR transgene. Data were acquired on an Attune NxT cytometer (Thermo). All data analysis was performed using FlowJo 9.0 software (FlowJo, LLC). The gating strategy can be found in Supplementary Fig. 5.
Heard A., Landmann J.H., Hansen A.R., Papadopolou A., Hsu Y.S., Selli M.E., Warrington J.M., Lattin J., Chang J., Ha H., Haug-Kroeper M., Doray B., Gill S., Ruella M., Hayer K.E., Weitzman M.D., Green A.M., Fluhrer R, & Singh N. (2022). Antigen glycosylation regulates efficacy of CAR T cells targeting CD19. Nature Communications, 13, 3367.
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7

Immunophenotyping of Lung Infiltrating Cells

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Lung infiltrating immune cells were stained with different combinations of fluorochrome-coupled antibodies against mouse CD45 (clone 30-F11, Biolegend), CD3 (clone 17A2, Biolegend), CD4 (clone GK1.5, Biolegend), CD8 (clone 53-6.7, Biolegend), CD11b (clone M1/70, Biolegend), CD11c (clone N418, Biolegend), Foxp3 (clone FJK-16s, eBioscience), CD279 (PD-1, clone 29F.1A12, Biolegend), CD152 (CTLA-4, clone UC10-4B9, eBioscience), Tim-3 (clone RMT3–23, eBioscience), CD223 (Lag-3, clone C9B7W, Biolegend), IL-2 (clone JES6-5H4, Biolegend), IFNγ (clone XMG1.2, Biolegend), BrdU (clone Bu20a, Biolegend). Jurkat, PD-1-Jurkat, and human PBMCs were stained with fluorochrome-coupled antibodies against human CD3 (clone HIT3a, Biolegend), CD4 (clone OKT4, Biolegend), and CD279 (PD-1, clone EH12.2H7, Biolegend).
Deng J., Wang E.S., Jenkins R.W., Li S., Dries R., Yates K., Chhabra S., Huang W., Liu H., Aref A.R., Ivanova E., Paweletz C.P., Bowden M., Zhou C.W., Herter-Sprie G.S., Sorrentino J.A., Bisi J.E., Lizotte P.H., Merlino A.A., Quinn M.M., Bufe L.E., Yang A., Zhang Y., Zhang H., Gao P., Chen T., Cavanaugh M.E., Rode A.J., Haines E., Roberts P.J., Strum J.C., Richards W.G., Lorch J.H., Parangi S., Gunda V., Boland G.M., Bueno R., Palakurthi S., Freeman G.J., Ritz J., Haining W.N., Sharpless N.E., Arthanari H., Shapiro G.I., Barbie D.A., Gray N.S, & Wong K.K. (2017). CDK4/6 Inhibition Augments Anti-Tumor Immunity by Enhancing T Cell Activation. Cancer discovery, 8(2), 216-233.
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8

Activation of PBMC Signaling

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Viable, frozen RM PBMCs from healthy donors were thawed, counted, and rested for 2 hours at 37°C, 5% CO2, at a concentration of 2 × 106 PBMCs per milliliter in vented cap bottles with RPMI 1640 (MilliporeSigma) supplemented with 5 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES; Corning), 2 mM glutamine (UCSF Cell Culture Facility), 50 μg/mL penicillin/streptomycin (Corning), 5 mM sodium pyruvate (Corning), and 10% FBS (Gibco). Upon resting, 1 × 106 PBMCs were transferred to a 48-well plate and left unstimulated or stimulated with IL-10 (5 ng/mL; catalog 200-10, PeproTech) with or without anti–IL-10 (MK-1966/JES3.12G8, Merck). To evaluate IL-10 signaling through phosphorylation of STAT3 (p-STAT3; clone 4/P-STAT3, BD Biosciences), different doses of anti–IL-10 were used (0.01–100 μg/mL). Frequencies of p-STAT3+ cells were evaluated after 30 minutes of stimulation by flow cytometry. The expression of Bcl-6 (a Tfh cell transcription factor; clone K112-91, BD Biosciences) or ICR markers (PD-1, clone EH12.2H7, BioLegend; and CTLA-4, clone BNI3, BD Biosciences) was evaluated by flow cytometry after 48-hour stimulation.
Harper J., Ribeiro S.P., Chan C.N., Aid M., Deleage C., Micci L., Pino M., Cervasi B., Raghunathan G., Rimmer E., Ayanoglu G., Wu G., Shenvi N., Barnard R.J., Del Prete G.Q., Busman-Sahay K., Silvestri G., Kulpa D.A., Bosinger S.E., Easley K.A., Howell B.J., Gorman D., Hazuda D.J., Estes J.D., Sekaly R.P, & Paiardini M. (2022). Interleukin-10 contributes to reservoir establishment and persistence in SIV-infected macaques treated with antiretroviral therapy. The Journal of Clinical Investigation, 132(8), e155251.
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9

Comprehensive Immune Cell Profiling

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Cell staining of whole blood was performed for 25 min on ice in the dark in staining buffer composed of PBS, 0.5% BSA, and 0.05% sodium azide. Red blood cells were lysed by addition of 1× BD Pharm Lyse Buffer (BD Biosciences) and incubation in the dark at room temperature for 15 min. Following one wash, cells were fixed in 2% paraformaldehyde for 15 min at room temperature, washed again, and resuspended in staining buffer. A minimum of 300,000 total events were collected on a FACS Calibur using Cell Quest and analyzed with FlowJo software (TreeStar). Anti-human CD20 (clone 2H7, catalog #302304), CD19 (clone HIB19, catalog #302234), CD38 (clone HB7, catalog #356608), CD3 (clone UCHT1, catalog #300426), CD4 (clone RPA-T3, catalog #300506), CD25 (clone BC96, catalog #302632), CD127 (clone A019D5, catalog #351325), CCR4 (clone TG6, catalog #335405), CXCR5 (clone TG2, catalog #335001), CD45RO (clone UCHL1, catalog #304218), and PD-1 (clone EH12.2.H7, catalog #329907) antibodies were purchased from BioLegend, anti-human CD27 (clone O323, catalog #12-0279-42) from eBioscience, and anti-human ICOS antibody (clone DX29, catalog #557802) from BD Biosciences.
Panwar B., Schmiedel B.J., Liang S., White B., Rodriguez E., Kalunian K., McKnight A.J., Soloff R., Seumois G., Vijayanand P, & Ay F. (2021). Multi–cell type gene coexpression network analysis reveals coordinated interferon response and cross–cell type correlations in systemic lupus erythematosus. Genome Research, 31(4), 659-676.
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