For cytokine restimulated human and mouse T cells, in some instances cells grown in normal media or tumor supernatant were treated with cycloheximide for 30 minutes prior to being restimulated for 4 hours with Cell Stimulation Cocktail and GolgiPlug Brefeldin A (eBioscience). For tumor infiltrating T cell analysis, single cell suspensions were counted and plated at 5x105 cells per well and stimulated with Cell Stimulation Cocktail and GolgiPlug Brefeldin A for 3 hours at 37C. Samples were then stained with Zombie NIR viability dye (Biolegend) followed CD45.2-BV510 (Biolegend, RRID:AB_2561393) and CD8-Alexa Fluor 647. IFNγ-PE (Invitrogen, RRID:AB_466192) and TNF-α-FITC (Invitrogen, RRID:AB_465418) intracellular FACS staining was performed using the Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent kit and Permeabilization Buffer (eBioscience). Samples were run on a BD Accuri C6 flow cytometer or a Cytek Northern Lights and analysis was performed with FlowJo software.
Ifn γ pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
IFN-γ-PE is a fluorescent-labeled antibody used to detect and quantify the presence of interferon-gamma (IFN-γ) in biological samples. It is a tool for researchers to measure IFN-γ expression in various cell types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 fitc, by Thermo Fisher Scientific (9 mentions)
Cd86 pe, by Thermo Fisher Scientific (4 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (3 mentions)
Foxp3 pe, by Thermo Fisher Scientific (3 mentions)
Il 4 pe, by Thermo Fisher Scientific (3 mentions)
Golgiplug, by BD (3 mentions)
Il 17 pe, by Thermo Fisher Scientific (3 mentions)
Cd3 efluor450, by Thermo Fisher Scientific (3 mentions)
Cd4 percp, by Thermo Fisher Scientific (3 mentions)
Cd25 pe, by Thermo Fisher Scientific (3 mentions)
Cd11c pe, by Thermo Fisher Scientific (3 mentions)
Cd4 apc, by Thermo Fisher Scientific (2 mentions)
Cd4 percp cy5, by BioLegend (2 mentions)
Foxp3 fitc, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Il 2 apc, by Thermo Fisher Scientific (2 mentions)
Cd25 apc, by Thermo Fisher Scientific (2 mentions)
Il 22 pe, by Thermo Fisher Scientific (2 mentions)
Cd19 percp, by BD (2 mentions)
Cd8 percp cy5, by Thermo Fisher Scientific (2 mentions)
32 protocols using ifn γ pe
1
Cytokine-Restimulated T Cell Analysis
2
Flow Cytometry Antibody Panel
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We purchased directly conjugated monoclonal antibodies for flow cytometry from Invitrogen (Waltham, MA, USA): CD4-PE, Clone GK1.5; CD11b-ef450, Clone M1/70; CD44-FITC, Clone IM7; CD45AF700, Clone 30-F11; CD206-APC, Clone MR6F3; FoxP3-ef450, Clone FJK-16S, IFN-γ-PE, Clone XMG1.2; GzmB-APC, Clone GB11; iNOS-PE, Clone CXNFT; live dead fixable dead cell stain kit, Catalogue No. L34959. BD Pharmingen (San Jose, CA, USA): CD3-AF700, Clone 17A2; CD4-Pacblue and perCPcy5.5, Clone RM4-5; CD8-FITC, perCPcy5.5, and PECY7, Clone 53-6.7; CD62L-PECY7, Clone MEL-14; H2Kd-PE, Clone 17A2; Ly6G-PE and percpcy5.5, Clone 1A8; Ly6C-APCCY7, Clone- AL-21; TNF-α PECY7, Clone MP6-XT22. Biolegend (San Diego, CA, USA): CD3-PECY7 Clone 17A2; B220-percpcy5.5, Clone RA3-6B2. R&D Systems: CCR2-APC, Clone 475301; CCL2-APC, Clone 123616. eBioscience (San Diego, CA, USA): H2Kd/Dd-ef450 Clone 34-1-25; CD4-APC, Clone GK1.5.
3
Isolation and Characterization of Retinal and CDLN Cells
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Cells were isolated from the retina and CDLNs on day 14 after immunization. Dead cells were excluded using live/dead dye (#423105, BioLegend, San Diego, CA, USA). Then they were stained with the following antibodies: the surface markers included: CD4 Percp-Cy5.5 (#100434), CD45 Brilliant Violet 605 (#103155) (BioLegend), TGFBR2 PE (#FAB532P, R&D Systems). For intracellular cytokine staining, the cells were stimulated with 5 ng/mL of phorbol myristate acetate, 500 ng/mL ionomycin, and 1 mg/mL brefeldin A (Sigma) at 37 °C for 5 h, following by fixation and permeabilization for 30 min. Then, cells were stained with antibodies detecting: IFN-γ PE (#505808), IL-17A Alexa Fluor 647 (#506912), FOXP3 FITC (#11-5773-82), Id2 PE-Cy7 (#25-9475-82, Invitrogen). For the Pim1 staining, cells were stained with surface antibodies, fixed, permeabilized, stained with Pim1 antibody (#3247S), then stained with Alexa Fluor 488-labeled antibody (#4412S) (Cell Signaling Technology, Danvers, USA). Finally, the cells were kept overnight at 4 ◦C and measured by flow cytometry. The flow cytometer (BD LSRFortessa, USA) was used for analysis and the results were analyzed with FlowJo software (version 10.0.7, Tree Star, Ashland, OR, USA).
4
Phenotypic and Functional Analysis of CD4 T Cells
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For phenotypic analysis of CD4 T cells, PBMCs were stained with CD4-FITC, CD8-PE, CD57-APC (BioLegend, San Diego, CA), CD28-PE (Invitrogen, Carlsbad, CA), CD45RA-PerCP710, PD1-FITC (eBioscience, San Diego, CA), or isotype control antibodies. To quantify cell apoptosis, PBMCs were stained with CD4-A647 and CD45RA-FITC for naïve or memory cell populations, and then stained with Annexin V-PE and 7-AAD using BD Pharmingen™ PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA). CD4+ T cells were also stained for caspase-3 expression following a protocol from CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Invitrogen). Levels of reactive oxygen species (ROS) in CD4 T cells were measured using the DCFDA-based Cellular ROS Detection Kit (Abcam, Cambridge, MA) or CellROX Green ROS Detection kit (ThermoFisher Scientific, Waltham, MA) according to manufacturer's protocol. For intracellular staining, the cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience), and stained with pATM (Ser1981)-PE antibody (BioLegend), pCHK2 (Thr68)-PE antibody, γH2AX-PE (eBioscience), IL-2-PE, and IFN-γ-PE (Invitrogen). Flow cytometry was carried out as described previously (19 (link), 20 (link)).
5
Flow Cytometric Analysis of T Cell Subsets
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To determine the Th1, Th2, Th17, and Treg cells in mice, flow cytometric analysis was performed on isolated DRG cells using CD4, Foxp3, IL-17A, TGF-β, and IL-4 antibodies as reported previously (18 (link)). Briefly, the cells suspension was transferred into 1 mL phosphate buffer saline (PBS) and centrifuged at 350 g for 5 min. After centrifugation, the supernatant was removed and the cells were resuspended with 500 μL fixation/permeabilization then centrifuged at 350 g for 5 mice after standing at room temperature for 30 min. The resuspension was repeated for twice. The cells were then incubated with monoclonal antibodies, including CD4-FITC, FOXP3-PE, IL-17A-PE, IL-4-PE, and IFN-γ-PE antibodies (eBiosciences, San Diego, California, USA) at dark for 30 min. After washing with PBS, the cells were resuspended in 150 μL PBS and then tested by using Beckman counter flow cytometer (USA). The data were analyzed using FlowjoX software. The lymphocytes were gated by FSC and SSC. CD4+IL-17A+, CD4+IL-4+, CD4+ IFN-γ+, and CD4+ FOXP3+ lymphocytes were identified as Th17, Th2, Th1, and Treg respectively.
6
Ex vivo 5T4 peptide stimulation
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Mouse PBMCs or splenocytes were stimulated ex vivo with 5T4 peptide pools (5 μg/ml of each peptide) and 1 μg/ml Golgi-Plug (BD) for 6 hours. Following stimulation, Fc receptors were blocked with anti mouse CD16/32 and cells were labelled with anti-mouse CD4-AlexaFluor700, and CD8-PerCPCy5.5 (eBioscience Ltd), fixed-permeabilized in CytofixCytoperm buffer (BD) and incubated with IFN-γ-PE, IL-2-APC and TNF-α-FITC antibodies (eBioscience Ltd).
To confirm 5T4 expression on the surface of the B16.h5T4 cell line, cells were gently dissociated and incubated with anti 5T4 antibody (clone H8, kind gift from Richard Harrop, Oxford BioMedica Ltd.) or mouse IgG isotype control antibody. Anti mouse IgG-FITC was used as secondary antibody.
All sample acquisitions were performed on a BD LSRII™ analyzer and data analysed with FlowJo software (Treestar).
To confirm 5T4 expression on the surface of the B16.h5T4 cell line, cells were gently dissociated and incubated with anti 5T4 antibody (clone H8, kind gift from Richard Harrop, Oxford BioMedica Ltd.) or mouse IgG isotype control antibody. Anti mouse IgG-FITC was used as secondary antibody.
All sample acquisitions were performed on a BD LSRII™ analyzer and data analysed with FlowJo software (Treestar).
7
Comprehensive Immunophenotyping of T and B Cells
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Phenotypic analyses of T cells and B cells were performed with anti-human monoclonal antibodies (mAbs): anti-human CD3-PerCP, CD4-FITC, CD19-PerCP and CD21-APC were from BD Biosciences (San Jose, CA, USA). CXCR5-APC, ICOS-PE, PD-1-PE, IFN-γ-PE, IL-4-PE, IL-17-PE, IL-21-PE, IL-22-PE, CD27-FITC, CD86-PE, CD95-PE, CD25-APC, and Foxp3-PE antibodies were obtained from eBiosciences (San Diego, CA, USA). Cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA) and data was analyzed by FlowJo software (Tree Star, San Carlos, CA).
8
Immune Cell Analysis Protocol
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The immune cells were analyzed following the standard protocol. Briefly, tumors were collected and incubated in dissociation buffer with 1640 medium (contained collagenase, hyaluronidase, and deoxyribonuclease I) at 37 °C for digest tumor tissues to the single‐cell suspension. And then the cells were stained with surface antibodies: CD3‐PE (eBioscience, Catalog: CD0304), CD4‐FITC (eBioscience, Catalog: 11‐0041‐82), CD8‐PerCP‐Cy5.5 (eBioscience, Catalog: 45‐0081‐82), CD86‐PE (eBioscience, Catalog: 12‐0862‐81), CD11b‐FITC (eBioscience, Catalog: 11‐0112‐81), F4/80‐PE‐Cy5 (eBioscience, Catalog: 15‐4801‐82), CD206‐PE (eBioscience, Catalog: 12‐2061‐82), CD44‐PE (eBioscience, Catalog: 12‐0441‐81), or CD62L‐APC (eBioscience, Catalog: 17‐0621‐81) respectively for 30 min. After fixing and perforating the cells, the intracellular markers: FOXP3‐PE (eBioscience, Catalog: 12‐5773‐82) or IFN‐γ‐PE (eBioscience, Catalog: 12‐7311‐82) were stained. The stained cells were detected using flow cytometry (Beckman Cytoflex S flow cytometer). The data were analyzed using FlowJo 10.0.
9
Multiparametric Flow Cytometry Immunoprofiling
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The following anti-mouse mAbs were used for surface staining during flow cytometry: CD3-eFluor 450, CD8-PerCP-Cy5.5, CD11b-AlexaFluor 700, CD11b-eFluor 450, CD14-FITC, CD25-APC, CD25-eFluor 450, CD45.1-APC, Ly6C-APC-eFluor 780, I-A/E-FITC (eBioscience, San Diego, CA, USA), CD45.1-eFluor 450, CD45R-Horizont V500, CD3-Horizont V500, CD4-Horizont V500, CD4-PerCP, CD8-Horizont V500, Ly6G-AlexaFluor 700, Siglec F-PE (BD Biosciences, San Jose, California, USA) and TLR4-APC (BioLegend, San Diego, Ca, USA). For intracellular staining, the following anti-mouse mAbs were used: TNFα-PE, Foxp3- and IFNγ-PE (eBioscience; San Diego, California, USA). Fc-block (anti-CD16/CD32 mAbs; eBioscience, San Diego, California, USA) was used both in surface and intracellular staining. For ELISA, matched anti-mouse capture mAb and biotinylated detection mAb against TNF-α, IFN-γ, IL-1β, IL-12 and IL-6 were used (R&D System, Minneapolis, Minnesota, USA). Blocking anti-mouse CD25 (PC61.5), CD4 (GK1.5) and anti-IFN-γ (XMG1.2) mAbs, as well as anti-mIL-2 mAbs for preparing IL-2 complexes (S4B6, JES6-1A12), were obtained from BioXcell (Lebanon, New Hampshire, USA).
10
Multiparameter Immune Cell Profiling
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CD3-percp-cy5.5 (Catalogue #45-0036-42), CD8-APC (Catalogue #17-0086-42), CD8-FITC (Catalogue #11-0086-42), CD14-APC (Catalogue #17-0149-42), CD56-FITC (Catalogue #4278380), CD16-PerCP-eFluor™710 (Catalogue #46-0168-42), CD11b-PerCP-eFluor™710 (Catalogue #46-0110-80), IFN-γ-PE (Catalogue #12-7319-42), TLR2-FITC (Catalogue #11-9922-41) and Mouse IgG1-PE (Catalogue #12-4714-81) were all bought from eBioscience. HLA-DR-PE (Catalogue #555812), IL-10-APC (Catalogue #554707), CXCR3-APC (Catalogue #550967) and IFN-γ-FITC (Catalogue #561053), Rat IgG2a-APC (Catalogue #554690) and mouse IgG-APC (Catalogue #5065947) were purchased from BD Biosciences. CD16-FITC (Catalogue #302006), HLA-DR-APC (Catalogue #307609), CD3-FITC (Catalogue #317306) and TLR4-APC (Catalogue #312815) were purchased from Biolegend. EP2-PE (Catalogue #10477) and Rabbit IgG-PE (Catalogue D5-1610) were purchased from Cayman Chemical.
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