Six-week-old female C57BL/6N mice were purchased from Japan SLC (Shizuoka, Japan) and kept in a specific pathogen-free environment. The animal use proposal and experimental protocols were reviewed and approved by The University of Tokyo Animal Care and Use Committee (ID:P15-125) and all animal procedures were conducted in accordance with institutional guidelines. The YTN2 and YTN16 cell lines [6 (link)] were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Kyoto, Japan) with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 100 μg/mL streptomycin, 100 U/mL penicillin (Nacalai Tesque), and MITO+ serum extender (Corning, Corning, NY, USA). Anti-PD-1 (RMP1-14), PD-L1 (10F9G2), CTLA-4 (9H10), and CD8α (53–6.7) mAbs were from Bio X Cell (Lebanon, NH, USA). DimerXI:Recombinant Soluble Dimeric Mouse H-2Db:Ig (H-2Db dimer), DimerXI:Recombinant Soluble Dimeric Mouse H-2Kb:Ig (H-2Kb dimer) were from BD Biosciences (Franklin Lakes, NJ, USA). FITC-conjugated anti-CD3ε, PerCP/Cyanine5.5-conjugated anti-CD4, APC/Cyanine7-conjugated anti-CD8, APC-conjugated anti-IFN-γ and Pacific Blue-conjugated anti-CD45 mAbs were from BioLegend (San Diego, CA, USA).
Apc conjugated anti ifn γ
Manufactured by BioLegend
Sourced in United States
APC-conjugated anti-IFN-γ is a monoclonal antibody that binds to the cytokine interferon-gamma (IFN-γ). The antibody is conjugated with allophycocyanin (APC), a fluorescent dye, allowing for the detection and quantification of IFN-γ-producing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol 12 myristate 13 acetate pma, by Merck Group (3 mentions)
Percp cy7 conjugated anti gzmb, by BioLegend (3 mentions)
Facscanto 2, by BD (2 mentions)
Pe conjugated anti tnf α, by BioLegend (2 mentions)
Apc cy7 conjugated anti cd8a, by BioLegend (2 mentions)
Ifnγ and tnfα secretion assay, by BioLegend (2 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Percp cyanine5.5 conjugated anti cd4, by BioLegend (1 mentions)
Mito serum extender, by Corning (1 mentions)
Dulbecco s modified eagle s medium dmem, by Nacalai Tesque (1 mentions)
C57bl 6n mice, by Japan SLC (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Celltrace violet reagent, by Thermo Fisher Scientific (1 mentions)
Apc cy7 conjugated anti cd3, by BioLegend (1 mentions)
Pe conjugated anti pd l1, by BioLegend (1 mentions)
Pe cy7 conjugated anti cd8, by BioLegend (1 mentions)
Ionomycin, by BD (1 mentions)
9 protocols using apc conjugated anti ifn γ
1
Investigating Antitumor Immunity in Mice
2
Assessing CD8+ T Cell Proliferation, Apoptosis, and IFN-γ Production
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To detect cell proliferation, sorted CD8+ T cells were labelled with CellTrace™ Violet reagent (5 mM; Life Technologies) in PBS and incubated for 15 min at 37 °C after treatment with siRNA for 6 h. Labelled CD8+ T cells were stimulated for 4 days with Phytohemagglutinin (PHA, 5 μg/ml, Sigma) [30 (link)]. Proliferation was measured at day 4 using a BD LSRII flow cytometer (Becton–Dickinson). To detect apoptosis, sorted CD8+ T cells from HCs were stimulated for 48 h with PHA after treatment with siRNA for 6 h. The cells were then stained with PE-conjugated-Annexin V and 7-aminoactinomycin D (7-AAD, Biolegend) for 30 min at 4 °C and analysed using an LSRII flow cytometer. An intracellular IFN-γ stimulation assay, was carried out in which freshly isolated CD8+ T cells were transfected with siRNA for 48 h and then stimulated for 6 h with 2 µl/ml Cell Activation Cocktail (each vial of this cocktail contained phorbol-12-myristate 13-acetate 40.5 µM, ionomycin 669.3 µM, and Brefeldin A 2.5 mg/ml in dimethylsulfoxide (DMSO) Biolegend). Finally, the cells were intracellularly stained with APC-conjugated anti-IFN-γ (Biolegend) and analysed using an LSRII flow cytometer.
3
Multiparametric Flow Cytometry Analysis
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Cells were stained with PE-conjugated anti-PD-L1, PerCp-conjugated anti-7-AAD, APC-Cy7-conjugated anti-CD3, PE-Cy7-conjugated anti-CD8, APC-conjugated anti-IFN-γ, and FITC-conjugated anti-granzyme B antibodies (BioLegend, USA). Dead cells were stained using 7-AAD. Among them, IFN-γ and granzyme B were used for intracellular staining as follows: cells were first fixed with 2% paraformaldehyde and permeabilized with 0.1% saponin in phosphate buffered saline (PBS) buffer. Next, cells were incubated in the dark for 15 min on ice with antibodies labeled with fluorochrome. For surface assessment, cells were incubated with fluorochrome-labeled antibodies directly. The cell phenotype was determined using cytofluorimetric analysis by flow cytometer (BD FACSCanto II, USA).
4
Profiling Cytokine Secretion in Tumor Infiltrating CD8+ T Cells
5
Multicolor Flow Cytometry Analysis of CNS-Infiltrating Immune Cells
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Single‐cell suspensions from the LN, spleen, and the CNS of mice were stained with extracellular fluorochrome‐conjugated CD antibodies (Biolegend), along with APC‐conjugated MOG38‐49 I‐Ab tetramer (NIH tetramer core, peptide sequence: GWYRSPFSRVVH) and Brilliant Violet 421‐conjugated human CLIP87‐101 I‐Ab tetramer as a control (peptide sequence: PVSKMRMATPLLMQA). Then, cells were fixed and permeabilized using the kit (eBioscience, Cat# 00‐5523‐00) for intracellular staining of PE‐conjugated anti‐FoxP3 (eBioscience Cat# 12‐5773‐82) per the instruction. For intracellular staining of CNS isolated immune cells, cells were stimulated with PMA (5 ng/ml), ionomycin (500 ng/ml), and protein transport inhibitor (0.7 µl/ml, BD Biosciences. Cat# 51‐2092KZ) for 5hrs, followed by extracellular marker and intracellular cytokine antibody staining. Data were analyzed using FlowJo™ v10 software. Mean fluorescence intensity (MFI) was defined as the “medians” of fluorescence intensities of the conjugated fluorochromes of the antibodies.
Cells isolated from the CP were incubated with PMA, ionomycin, and protein transport inhibitor as above for 5 h, followed by extracellular staining with PerCP/Cy5.5 conjugated anti‐CD45 (Biolegend, Cat# 103132) and intracellular staining with APC‐conjugated anti‐IFN‐γ (Biolegend, Cat# 505810).
Cells isolated from the CP were incubated with PMA, ionomycin, and protein transport inhibitor as above for 5 h, followed by extracellular staining with PerCP/Cy5.5 conjugated anti‐CD45 (Biolegend, Cat# 103132) and intracellular staining with APC‐conjugated anti‐IFN‐γ (Biolegend, Cat# 505810).
6
Multiparametric Flow Cytometry Analysis
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Fluorochrome-conjugated anti-CD3, CD4, CD8, CD44, CD25, and CD62L mAbs were purchased from BioLegend (CA, USA). Anti-CD98hc antibody was described previously [13 ]. APC-conjugated AnnexinV was purchased from BD Biosciences (NJ, USA). To detect intracellular expression of IFN-γ by flow cytometry, cells were stimulated with PMA (0.04 μM) and ionomycin (1.3 μM) for 5 hours in the presence of monensin (2 mM). Then, cells were stained with a PB-conjugated anti-CD4 mAb and fixed with 4% paraformaldehyde. After washing, cells were stained with APC-conjugated anti-IFN-γ (BioLegend) in a buffer containing saponin. Fluorescent signals were acquired with a FACS CantoII (BD Biosciences) and Flow-Jo software (Tree Star, Inc) was used for analysis.
7
Intracellular IFN-γ Expression in γδT Cells
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After 12–15 days’ culture, the intracellular IFN-γ expression assay was performed using flow cytometry. γδT cells were incubated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 250 ng/mL ionomycin, and monensin (6 μg/mL) for 4 h at 37°C in 5% CO2. Then, γδT cells were collected, and the following antibodies were used for intracellular staining: PE-Cy7-conjugated anti-CD3 (BioLegend), PE-conjugated anti-TCRγδ (BD Pharmingen), and APC-conjugated anti-IFN-γ (BioLegend). We used a Cytofix/Cytoperm Plus Kit (BD Pharmingen) for intracellular staining, according to the manufacturer’s protocols.
8
Quantifying IFN-γ and GZMB in Tumor Organoid CD8 T Cells
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After drug treatment, IFNγ and GZMB expression levels in CD8 T cells incorporated in tumor organoids were detected. Tumor organoids were digested into single cells by TrypLE Express at 37 °C shaking at 300 rpm for 15 min. The single cells including CD8 T cells were stimulated with 50 ng mL−1 phorbol 12‐myristate 13‐acetate (PMA) (Sigma‐Aldrich) and 1 µm ionomycin (Sigma‐Aldrich) for 4 h, in the presence of 5 µg mL−1 Brefeldin A, which can amplify intracellular cytokine staining signals.[64 ] The cells were stained with APC/Cy7‐conjugated anti‐CD8α (BioLegend) and later PerCP/Cy7‐conjugated anti‐GZMB and APC‐conjugated anti‐IFNγ (BioLegend) for intracellular staining. The stained cells were analyzed by flow cytometry.
9
Profiling Cytokine Secretion in Tumor Infiltrating CD8+ T Cells
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We measured cytokine and cytolytic granule production (IFNγ, GZMB and TNFα) of CD8+ T cells from mouse tumour samples and tumour organoid-T cell co-culture. Briefly, CD8+ T cells were placed into the 24-well plate at 1 × 106 cells/well, stimulated with 1 μM ionomycin and 50 ng ml−1 phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 4 h, in the presence of 5 μg ml−1 Brefeldin A (BFA), with the purpose to amplify the expression of intracellular cytokines61 (link). The cells were stained with APC/Cy7-conjugated anti-CD8a (Biolegend) for 15 min and then fixed by 4% PFA. After washing, cells were stained with PerCP/Cy7-conjugated anti-GZMB, APC-conjugated anti-IFNγ and PE-conjugated anti-TNFα (Biolegend) for 15 min. In flow cytometric analyses, T cells stained with isotype control antibodies were used as negative controls for gating the cytokine or granule-producing cells. For T cells from tumour organoid-T cell co-culture, tumour organoids were first digested into single cells with TrypLE Express at 37°C before APC/Cy7-conjugated anti-CD8a staining. In the IFNγ and TNFα secretion assay (Biolegend), T cells were stimulated with ionomycin and PMA without the presence of BFA. The media were collected for ELISA assay.
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