Single-cell suspensions of digested tumors from OPC and OPC-API mice (1 × 106 cells) were plated onto a 96 well plate coated with purified anti-mouse CD3 (10µg/mL) (Biolegend). Soluble purified anti-mouse CD28 (2µg/mL) (Biolegend) in RPMI 1640 media (10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) was added to the assay wells and then incubated at 37 °C in 5% CO2. After two days of incubation, Golgi Plug, containing brefeldin A, (1 ug/mL) (BD Bioscience) was added to each assay well and incubated for 4 hrs. After incubation, cells were surface stained with fluorescent antibodies including anti-mouse CD3-FITC (Biolegend) and anti-mouse CD8-PercP/Cy5.5 (Biolegend). Samples were then intracellularly stained with anti-mouse IFN-γ-PE (eBioscience), along with isotype CTRL, using a Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer’s protocol. Acquisition of activated CD8+ T cell samples were performed using a flow cytometer BD LSRII (BD Biosciences Immunocytometry Systems). FlowJo software (BD Bioscience) was used for data analysis.
Anti mouse ifn γ pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-mouse IFN-γ-PE is a fluorescently-labeled antibody that binds to mouse interferon-gamma (IFN-γ). It is designed for use in flow cytometry and other immunoassays to detect and quantify IFN-γ expression in mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Golgistop, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Anti mouse cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Fitc anti mouse cd3, by BioLegend (1 mentions)
Golgiplug containing brefeldin a, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Anti mouse cd3, by BioLegend (1 mentions)
Anti mouse cd8 percp cy5, by BioLegend (1 mentions)
Anti tgfβ pe, by R&D Systems (1 mentions)
Anti mouse il 10 pe, by Thermo Fisher Scientific (1 mentions)
Concanavalin a type 4 cona, by Merck Group (1 mentions)
Pe anti mouse cd11c, by BD (1 mentions)
Fitc anti mouse ifn γ, by BD (1 mentions)
D luciferin, by Promega (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Anti p stat3 s727 pe, by BD (1 mentions)
10 protocols using anti mouse ifn γ pe
1
CD8+ T Cell Activation Assay
2
Intracellular Cytokine Profiling by Flow Cytometry
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Cells required for intracellular cytokine staining were cultured for 6 h in RPMI 1640 containing PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich). GolgiStop (BD Biosciences) was added to the culture for the last 4 h. Cells were surface stained with anti–CD19-PeCy7 or Pacific Blue (BioLegend), anti–TGF-β–PE (R&D Systems), anti-CD11c FITC or allophycocyanin (eBioscience), or anti-F4/80 allophycocyanin (eBioscience). Cells were then washed and stained with anti-mouse IFN-γ PE, anti-mouse IL-17 Alexa Fluor 647 (eBioscience), or anti-mouse IL-10 PE and analyzed by flow cytometry. Where possible, all gates were set using isotype control Abs. All samples were run on the LSRII or LSR Fortessa and analyzed using FlowJo software (Tree Star, Ashland, OR). All Abs were purchased from BD Biosciences unless otherwise stated.
3
Immunophenotyping of Murine Immune Cells
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D-Luciferin was purchased from Promega (Madison, Wisconsin). Concanavalin A (type IV) (ConA) was obtained from Sigma-Aldrich Chemical Co (St. Louis, MO). Anti-mouse-TNF-α-PE, anti-mouse-IFN-γ-PE, anti-mouse-CD335(NKp46)-PE, anti-mouse-TRAIL-PE, anti-mouse-CD178(FasL)-PE, and anti-mouse-perforin-PE were obtained from eBioscience (San Diego, CA). Anti-mouse-CD134-PE, anti-mouse-CD69-PE, anti-mouse-IFN-γ-FITC, anti-mouse-CD11b-PE, anti-mouse-NK1.1-FITC, anti-mouse-CD11c-PE, and anti-mouse-CD3e-PE-CyTM5 were obtained from BD Biosciences (San Jose, California).
4
Cytometric Analysis of T Cell Subsets
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Stimulated T cells and IL-12 receptor-specific siRNA-transfected T cells were stained with anti-mouse CD4-peridinin-chlorophyll protein and anti-mouse CD25-allophycocyanin for 30 min at 4 °C. The cells were permeabilized and fixed with CytoPerm/CytoFix (BD Biosciences, San Diego, CA, USA) in accordance with the manufacturer’s protocol; further stained with anti-mouse Foxp3-phycoerythrin (PE) and/or anti-mouse IL-17-fluorescein isothiocyanate (FITC), anti-mouse IFN-γ-PE, and anti-mouse IL-4-PE (all from eBioscience, San Diego, CA, USA); and subjected to flow cytometric analysis (FACSCalibur; BD Biosciences). For p-STAT3 Y705 and S727 and p-STAT5 analysis, splenocytes were treated with p40-EBI3 (1 and 10 μg/mL) and IL-6 for 1 h; the cells were then stained with anti-mouse CD4-FITC. The cells were fixed and permeabilized with Lyse/Fix Buffer (BD Pharmingen, San Jose, CA, USA) and Perm Buffer II (BD Pharmingen). The permeabilized cells were stained with anti-p-STAT3 Y705-PE, anti-p-STAT3-S727-PE, or anti-p-STAT5-PE (BD Pharmingen) and subjected to flow cytometric analysis (CytoFLEX; Beckman Coulter, Fullerton, CA, USA).
5
Splenocyte IFN-γ Secretion Assay
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Mice were immunized as mentioned above. PBS-treated mice were used as the control. Spleens were excised from the mice 14 days after the first immunization. Splenocytes were separated and counted on a Guava easyCyte 8HT (Merck Millipore, Billerica, Massachusetts, USA) with Viacount reagent (Merck Millipore, Billerica, Massachusetts, USA). The densities of the splenocytes were adjusted to 2.0 × 106 cells/ml in complete RPMI 1640 containing 10% FBS. Splenocytes from each mouse were added to 2 wells of a 24-well plate (4.0 × 106 cells per well). For the tfRFP-stimulated groups, 50 μg/ml tfRFP was added to the medium. An equal volume of PBS was added to the control groups. The splenocytes were cultured at 37ºC for 3 days. IFN-γ secretion was inhibited by monensin (Biolegend, San Dieg, California, USA) for the last 4 h of the culture. Finally, collected cells were fixed and stained with anti-mouse CD3 APC/Cy7 (Biolegend, San Dieg, California, USA), anti-mouse CD4 APC (Biolegend, San Dieg, California, USA), anti-mouse CD8 PE/Cy7 (Biolegend, San Dieg, California, USA), and anti-mouse IFN-γ PE (eBioscience, San Diego, California, USA) for flow cytometric analysis. A rat anti-mouse IgG1 kappa PE antibody (eBioscience, San Diego, California, USA) was used as an isotype control.
6
Flow Cytometry Analysis of Immune Cells
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Dissected nerves were mechanically dissociated with a cell strainer (70 μm pores), and cells were collected in Petri dishes with cold PBS. To increase intracellular protein production, cells were treated for 3 h at 37°C with Phorbol 12-myristate 13-acetate (PMA, 50 ηg/mL, Sigma) and Ionomycin (500 ηg/mL). Brefeldin A (1 μg/mL) was added to prevent protein release by the cells. Samples were fixed and permeabilized with commercial buffers (Fixation/Permeabilization buffers, eBioscience, Cat. code: 00–5521) according to the manufacturer’s instructions, then washed with PBS, and incubated with antibodies as follows: anti-CD8/FITC, anti-IL-10/APC and anti-IFN-g/PE. 30,000 events were acquired from each sample in the flow cytometer (FACS Canto, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed in FlowJo 10.0.5 software (Tree Star Inc., Ashland, OR, USA). Gating for flow cytometry was performed using unstained cells and FMO (Fluorescence Minus One) analysis, as described elsewhere [16 (link)]. For cell staining, we used these antibodies: anti-mouse CD8a FITC (0.5 μg, eBioscience, Cat. code: 11–0081), anti-mouse IFN-γ PE (0.5 μg, eBioscience, Cat. code: 12–7311) and anti-mouse IL-10 APC (0.5 μg, eBioscience, Cat. code: 17–7101).
7
Multiparameter Flow Cytometry Analysis
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Anti-mouse CD11c-PE, anti-mouse MHC class II I-Ab-FITC, anti-mouse CD80-APC, anti-mouse CD86-APC, anti-mouse/rat CD40-FITC, anti-mouse CD4-FITC, anti-mouse IFN-γ-PE, and anti-mouse/rat IL17A-APC were purchased from eBioscience. Data were obtained using FACScanto or FACSAria III (BD Bioscience) and analyzed with the FlowJo software (Tree star, Ashland, OR).
8
Comprehensive Immunophenotyping of CD8+ T Cells
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Flow cytometry of CD8+ T cells was performed using a CytoFLEXS FACS (Beckman Coulter Life Sciences, Mississauga, Ontario, Canada) to detect IRs, cytokine levels and proliferation. Lymphocytes were stained with 0.2 µg of each of the following MAbs: FITC anti-mouse CD8, PerCP-eFluor 710 anti-mouse PD-1, PE-CY7 anti-mouse TIM-3, APC anti-mouse BTLA, PerCP-eFluor 710 Rat IgG2b Isotype Control, PE Mouse IgG2a κ Isotype Control, FITC Mouse IgG2a κ Isotype Control, PE-CY7 Mouse IgG2a κ Isotype Control, APC Mouse IgG1 κ Isotype Control, PE anti-mouse IL-2 PE anti-mouse TNF-α and PE anti-mouse IFN-γ- (eBioscience, San Diego, CA, USA). A Fixation/Permeabilization Solution Kit (BD Biosciences, USA) was used for intracellular staining where required for different experiments.
9
Comprehensive Inflammatory Cytokine Analysis
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Complete Freund’s adjuvant (CFA) was bought from Sigma (Shanghai, China). Sac/Val and valsartan were purchased from Novartis Pharma AG (Basel, Switzerland). Antibodies against IFN-γ (Cat# sc-8423, RRID: AB_627,778), IL-1β, IL-17 (Cat# sc-374218, RRID: AB_10988239), and β-actin were bought from Santa Cruz (Dallas, USA). Anti-IL-18 antibody was purchased from Arigobio (Hsinchu City, Taiwan). The IL-6 ELISA kit (Cat# M6000B, RRID: AB_2877063) and the hsCRP ELISA kit were purchased from R andD (Minneapolis, USA). The IFN-γ and IL-17 ELISA kits were purchased from Neobioscience (Shenzhen, China). The cTnT quantitative rapid assay kit and collagenase B were bought from Roche Diagnostics (Shanghai, China). The total protein extraction kit, super ECL reagent, and antibody against ROR-γt (Cat# 14-6988-80, RRID: AB_1311291) were bought from Thermo Fisher Scientific Inc. (Hanover Park, USA). Antibodies against NLRP3 (Cat# AG-20B-0014, RRID: AB_2490202), ASC (Cat# AG-25B-0006, RRID: AB_2490440) and caspase-1 (Cat# AG-20B-0042, RRID: AB_2490248) were purchased from AdipoGen. Antibodies against T-bet, sGC-α1, sGC-β1, total-NF-κB p65 and phospho-NF-κB p65 were purchased from ImmunoWay (Plano, United States). FITC-anti-mouse CD4, PE-anti-mouse IFN-γ and allophycocyanin-anti-mouse IL-17 were purchased from eBioscience.
10
Quantification of Th1 and Th17 Cells
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After stimulation for 5 days, splenocytes were harvested and stimulated with PMA (Sigma, 50 ng/mL), ionomycin (Sigma, 1 mM), and Golgistop (BD Biosciences, 2μl in 3mL culture medium) for another 6 h. Then, the splenocytes were incubated with FITC-anti-mouse CD3 (GK1.5), APC-anti-mouse CD4 (GK1.5), PE-anti-mouse IFN-γ (XMG1.2), and PerCP-Cyanine5.5-anti-mouse/rat IL-17A (eBio17B7) (eBioscience) in FACS buffer (1% BSA, 1% duck serum, and 0.01% sodium azide) at 4 °C for 30 min. The cells were then washed 3 times with PBS. The percentage of Th1 (IFN-γ+ IL-17A−) and Th17 (IFN-γ− IL-17A+) cells was evaluated using a FACSCalibur (BD Biosciences) and analysed using the CellQuest Pro software (BD Biosciences).
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