PE-conjugated HLA-A*0201 RMSAPSTGGV tetramer (H3.3K27M-tetramer) was produced by the National Institute of Allergy and Infectious Disease tetramer facility within the Emory University Vaccine Center (Atlanta, GA) using the peptide synthesized by A&A Laboratories. PE-conjugated HLA-A*0201/RMSAPSTGGV Dextramer was purchased from Immudex. Cells were stained with tetramer (10 µg/ml) or dextramer in PBS containing 1% BSA for 15 min at 4°C (for tetramer) or room temperature (for dextramer), followed by surface staining for various T cell markers at 4°C. Cells were then washed with PBS containing 0.1% BSA. For some experiments, T cells were stained with tetramer, followed by anti–human CD3 FITC (344803; BioLegend), CD4-PerCPCy.5.5 (317427; BioLegend), CD8 APC (344722; BioLegend), CD69 FITC (11-0699-42; eBioscience), or PD-1-PECy7 (561272; BD Biosciences) along with the suitable isotype control antibodies. Intracellular cytokine staining was performed using Fixation/Permeabilization Solution kit (54714; BD Biosciences) according to the manufacturer’s instructions. T cells were then stained with anti–human Granzyme-B-BV421 (563389; BD Biosciences). The cells were acquired using a Sony SH800 flow cytometer and analyzed using FlowJo software v.10.
Pd 1 pe cy7
Manufactured by BD
Sourced in United States
PD-1-PE-Cy7 is a fluorescently labeled antibody targeting the Programmed Cell Death-1 (PD-1) receptor. It is designed for flow cytometric analysis of cells expressing PD-1.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 bv785, by BioLegend (2 mentions)
Facsdiva software, by BD (2 mentions)
Tim 3 pe, by BD (2 mentions)
Cd3 apc h7, by BD (2 mentions)
Cd8 pb, by BD (2 mentions)
Cd4 pb, by BD (2 mentions)
Sh800 flow cytometer, by FlowJo (1 mentions)
Cd69 fitc, by Thermo Fisher Scientific (1 mentions)
Cd8 apc, by BioLegend (1 mentions)
Anti human cd3 fitc, by BioLegend (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Cd4 percp cy5, by BioLegend (1 mentions)
Cd4 v500, by BD (1 mentions)
Cd45ra bv 421, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Cd8 apc h7, by BD (1 mentions)
Cd4 fitc, by BioLegend (1 mentions)
Human fc block, by BD (1 mentions)
Anti human cd3 bv605, by BD (1 mentions)
Ccr7 percp cy5, by BD (1 mentions)
17 protocols using pd 1 pe cy7
1
Quantification of Antigen-Specific T Cells via HLA Tetramers
2
Comprehensive T-cell Immunophenotyping by Flow Cytometry
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For surface staining, cells were incubated at room temperature with human Fc block (BD Biosciences, San Diego, CA, USA) and followed by staining with directly conjugated mAbs for 30 min at 4 °C. The mAbs used were anti-human CD3-BV605, CD4-V500, CD8-APC-H7, CD45RA-BV421, CCR7-PerCp-Cy5.5, PD-1-PE-Cy7, CD160-Alexa Fluor 488 (BD Biosciences), CD4-FITC, TIM-3-BV421, 2B4-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA), and TIGIT-APC (eBioscience, San Diego, CA, USA). Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences). FlowJo Software (Tree Star, Ashland, OR, USA) was used in data analysis.
3
Phenotyping of Th cell subsets
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Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-treated whole blood samples by density gradient centrifugation using Lymphoprep ™ (Fresenius Kabi, Oslo, Norway).
Peripheral blood lymphocytes (PBL) were isolated and stained with the following fluorochrome-conjugated monoclonal antibodies: CD4-APC-H7, CXCR3-PE, CCR6-BB515, PD-1-PE-Cy7 and ICOS-BV450 (all from BD biosciences, San José, CA, USA) (Mallett et al., 2019 (link); Mousset et al., 2019 (link)). Acquisition of PBLs was performed with a FACS CANTO II flow cytometry (BDbiosciences) and analyzed with Flow-Jo software v10.6.2. Gating strategy is shown (Figure S1
Cells CXCR3+/CCR6- gated from CD4 were considered Th1 cells, CXCR3-/CCR6+ cells were considered as Th17 and CXCR3-/CCR6- as Th2. Regarding the activation grade, cells PD-1-/ICOS- were considered as quiescent Th cells, PD-1-/ICOS+ cells as early-activated cells, PD-1+/ICOS+ as late activation markers and PD-1+/ICOS- as exhausted or senescent cells (Mallett et al., 2019 (link)).
Peripheral blood lymphocytes (PBL) were isolated and stained with the following fluorochrome-conjugated monoclonal antibodies: CD4-APC-H7, CXCR3-PE, CCR6-BB515, PD-1-PE-Cy7 and ICOS-BV450 (all from BD biosciences, San José, CA, USA) (Mallett et al., 2019 (link); Mousset et al., 2019 (link)). Acquisition of PBLs was performed with a FACS CANTO II flow cytometry (BDbiosciences) and analyzed with Flow-Jo software v10.6.2. Gating strategy is shown (
Cells CXCR3+/CCR6- gated from CD4 were considered Th1 cells, CXCR3-/CCR6+ cells were considered as Th17 and CXCR3-/CCR6- as Th2. Regarding the activation grade, cells PD-1-/ICOS- were considered as quiescent Th cells, PD-1-/ICOS+ cells as early-activated cells, PD-1+/ICOS+ as late activation markers and PD-1+/ICOS- as exhausted or senescent cells (Mallett et al., 2019 (link)).
4
Multicolor Flow Cytometry of Immune Cells
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PBMC, TDLN and NTDLN single-cell suspensions were washed in FACS buffer (PBS+0.1% bovine serum albumin (BSA)+0.02% NaN3). Cells were resuspended in 50 μL of FACS buffer and stained in a total volume of 75 μL for 30 min at 4 °C for surface expression using monoclonal antibodies directed against CD3 (PerCP-Cy5.5), CD8 (V500), CD25 (APC), CD127 (BV421), CD45RA (APC-H7), PD-1 (PE-Cy7) (BD Biosciences, San Jose, CA, USA) and CD4 (AF700) (Biolegend, San Diego, CA, USA) pre-diluted in Brilliant-violet staining buffer (BD Biosciences). Cells were washed with cold PBS and fixated and permeabilised using the eBioscience FoxP3 staining kit according to the manufacturer's instructions. Antibodies against FoxP3 (PE) (eBioscience, San Diego, CA, USA) and Ki-67 (FITC) (BD Biosciences) were used for intracellular staining and incubated for 30 min at 4 °C. Stained cells were analysed on a BD LSR Fortessa X-20 flow cytometer, and data were analysed using Kaluza analysis software (Beckman Coulter, Brea, CA, USA). Mean fluorescence intensity values calculated by Kaluza were multiplied by ten to be equivalent to the values measured with the Diva software on the LSR Fortessa X-20.
5
Comprehensive Immune Cell Profiling
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Fluorochrome-conjugated anti-mouse monoclonal antibodies CD3-AF700 (cat # 100216), CD4-BV785 (cat # 100453), CD8-PB (cat # 100725), CD25-BV650 (cat # 102038), GITR-PECy7 (cat # 120222), ICOS-PECy5 (cat # 107708), IFNg-PeCy7 (cat # 505826), and IL-2-PB (cat # 503820) were purchased from Biolegend. CD44-Percp-cyanine5.5 cat# 45-0441-80, CD62L-APCeFL780 (cat # 47-0621-82), Foxp3-APC (cat# 17-5773-82), Eos-eFL660 (cat # 50-5758-80), Helios-PeCy7 (cat # 25-9883-42), Aiolos-PE (cat # 12-5789-80),and bCatenin-eFL660 (cat # 50-2567-42) were purchased from Thermo Fisher Scientific. PD-1-PECy7 (cat# 25-9985-80), CXCR5-BV421 (cat # 562889), Bcl-6-PE (cat # 569522), and phospho-STAT5-PE (pY694) (cat # 612567) were procured from BD Biosciences.
6
Multiparametric Immune Phenotyping of Cells
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Cells were thawed and cultured in AIM-V culture medium with 5% heat-inactivated human serum in 24-well plates at a concentration of 3–5 × 106 cells/ml. Plates were incubated in a humidified incubator at 37°C, with 5% CO2. After 48 hours of resting, cells were harvested and washed in FACS buffer before surface staining. For exhaustion profile analysis, cells were stained at 4°C for 30 min., washed, and, for tubes only intended for surface staining, resuspended in FACS buffer. For tubes intended for intracellular staining, cells were permeabilized using BD Bioscience Cytofix/CytopermTM Kit according to manufacturer's instructions. The following antibodies were used: CD4-PerCP, CD57-FITC, CD27-PE, CD56-PE-Cy7, CD28-APC, CD8-AmCyan, ICOS-PE, BTLA-PE, CTLA-4-APC, PD-1-PE-Cy7 (all from BD Bioscience, San Jose, CA, USA), Near Infra Red dead cell marker (Dako), LAG-3-FITC (LifeSpan Biosciences), TIM-3 (eBioscience), CD107a; PECy7-conjugated IFN-c; PerCP-conjugated CD8; APC-conjugated CD4, TNF-a; APCCy7-conjugated CD3. Cells were resuspended in FACS buffer prior to acquisition, and the cells were acquired using a BD FACSCanto II flow cytometer. A minimum of 100 000 events were recorded per sample. Analysis was performed with the BD FACSDiva Software (BD Bioscience,)
7
Phenotyping T Cell Subsets by Flow Cytometry
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Flow cytometry (FACS Calibur, BD, USA) was used to measure CD4 + and CD8 + T cell counts immediately after PVB isolation. PBMCs were isolated from PVB using Ficoll density gradient centrifugation in the laboratory following the manufacturer's instructions. The following cocktail of antibodies was used to stain PBMCs immediately after isolation to avoid the loss of immune epitopes caused by freeze–thaw: CD3 APC-H7, CD4 FITC, CD8 APC, CD45RA BV711, CCR7 BV650, PD-1 PE-Cy7, Tim-3 PE, and FVS510 AmCyan (all from BD Biosciences, USA). Within 24 h, cell-surface receptor expression was quantified by flow cytometry (FACS LSRFortess, BD, USA). At least 105 cell populations were acquired as gated events and collated for each sample. Subsets analysis was conducted using Flowjo software (version 10.0, BD, USA). Appropriate isotype-matched controls or fluorescence minus ones were run in parallel for each sample.
8
Multiparametric Phenotyping of Antigen-Specific T Cells
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Frozen PBMC were thawed and stained ex vivo with HLA*0201 or HLA*0101 PE-labelled pentamers (Proimmune Oxford; 20 min in PBS, RT), stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher; 20 min RT), fixed (1% formaldehyde in PBS, 20 mins RT) then stained with: CD3-PO (Pacific Orange; Invitrogen, UCHT1), CD8–PB (Pacific Blue; BD biosciences, RPA-T8), and either stained with CCR7-PeCy7 (CD197; BD biosciences, 3D12), CD45RA-FITC (BD biosciences, HI100), CD38-PerCP-Cy5.5 (Biolegend, HIT2), HLA-DR-AlexaFlour700 (BD biosciences, G46-6) for 30 min RT; or permeabilised (10x permeabilisation buffer; ebiosciences) and stained with PD1-PeCy7 (BD biosciences, EH12.1), Perforin-FITC (BE biosciences, dG9), Granzyme B- Alexa fluor700 (BD biosciences, GB11) and Granzyme A-PerCpCy5.5 (Biolegend, CB9) 30 min RT. For intranuclear staining, PBMC were stained as above for pentamers and Live/Dead dye, and then surface stained with: CD3-Pacific-orange, CD8-PB (30 min RT). PBMC were then fixed (1 h RT) and permeabilised using the Foxp3/Transcription Factor Staining Buffer Set (ebiosciences; 45 min RT) and stained with eomes-eFluor660 (eBiosciences, WD1928) and Tbet-BV605 (Biolegend, 4B10).
9
Comprehensive Murine and Human Immune Profiling
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Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).
10
Comprehensive Immunophenotyping Protocol
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Chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse or anti-human fluorochrome labeled antibodies (Abs): CD4-PB, CD62L-APC, CD44-PerCP-Cy5.5, CD127-PE, FoxP3-FITC, Tim3-PE, PD1-PECy7, IFN-γ-PECy7, TNFα-PerCPCy5.5, IL-17-PerCPCy5.5, CD25APC-Cy7, CD45RA-PE, CD45RO-APC, and Abs for ELISA were procured from BD Pharmingen (San Diego, CA) or otherwise mentioned. RPMI-1640 and FBS were purchased from GIBCO (Grand Island, NY) for cell culture. For culturing of cells, tissue culture grade plastic-wares were purchased from BD Biosciences (Bedford, MA). Ab against iNOS used in Western blot was procured from (Abcam, Cambridge, United Kingdom).
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