During the last 4 or 16 hours of culture depending upon the experiment, Brefeldin A (3 μg/ml) and Golgistop (1/2000 final dilution; both from BD Biosciences, Mountain View, CA) were added to block cytokine secretion. For diluted whole blood cultures, red cells were lysed with 5 volumes of 1x PharmLyse solution (BD Biosciences, Oxford, UK) for 10 minutes at room temperature. Labeling of dead cells, fixation and permeabilization were performed as previously described [24 (link)]. Depending upon the experiment, cells were surface stained with anti-CD4-APC-H7 (BD Biosciences), anti-CD19-efluor450 and anti-CD14-efluor450 (eBiosciences) for 30 minutes at 4°C, or, following permeabilisation, with anti-CD3-Horizon-V500, anti-IL-2-FITC, anti-TNFα-PE-Cy7 (BD Biosciences), anti-IL-17-efluor660, and anti-IFNγ-PerCP-Cy5.5 (Biolegend) for 30 minutes at room temperature. Cells were finally resuspended in 250 μL 1% paraformaldehyde (Sigma, UK) and filtered prior to acquisition on a FACS Canto II flow cytometer or an LSRII flow cytometer (BD Biosciences). Compensation was performed using tubes of CompBeads (BD Biosciences) individually stained with each fluorophor and compensation matrices were calculated with FACSdiva. Data were analyzed using FlowJo software version 9 (Treestar, Ashland, OR). Gating strategy and an example of raw flow cytometry data is shown in S1 Fig .
Anti ifnγ percp cy5
Manufactured by BioLegend
Sourced in United States
Anti-IFNγ-PerCP-Cy5.5 is a fluorescently labeled antibody that binds to and detects interferon-gamma (IFNγ). It is designed for use in flow cytometry and other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Anti tnf α fitc, by BioLegend (2 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Pharm lyse solution, by BD (1 mentions)
Anti cd4 apc h7, by BD (1 mentions)
Compbead, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Anti cd19 efluor450, by Thermo Fisher Scientific (1 mentions)
Anti cd3 horizon v500, by BD (1 mentions)
Brefeldin a, by BD (1 mentions)
Anti il 2 fitc, by BD (1 mentions)
Anti tnfα pecy7, by BD (1 mentions)
Anti cd3 pe, by BD (1 mentions)
Anti tnf α apc, by BioLegend (1 mentions)
Anti cd56 pe cf594, by BD (1 mentions)
Anti cd107a pe cy7, by BD (1 mentions)
Anti cd16 fitc, by BD (1 mentions)
Anti cd4 apc, by BioLegend (1 mentions)
Anti il 2 pe, by BioLegend (1 mentions)
6 protocols using anti ifnγ percp cy5
1
Cytokine Production Assay Protocol
2
Cytokine Production and Degranulation of NK Cells
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IFN-γ and TNF-α production by NK cells was evaluated in all samples available for phenotypic analysis after overnight stimulation with or without IL-12 and IL-18 (5 ng/mL) in the presence of 10 µg/mL of brefeldin A (BFA) added during the last 3 h of culture. Surface staining with anti-CD3-PE, anti-CD56-PE-CF594, and anti-CD16-FITC (BD Bioscience, Franklin Lakes, NJ, USA) was performed. Then, cells were fixed with medium A reagent and permeabilized with medium B reagent (Nordic Mubio, Lifespan Biosciences, Seattle, WA, USA) in accordance with manufacturer’s instructions. Cytokine determinations were performed by intracellular cytokine staining (ICS) with anti-IFN-γ-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA) and anti-TNF-α-APC (BioLegend, San Diego, CA, USA) monoclonal antibodies and analyzed by flow cytometry. The CD107a degranulation assay was performed to assess the cytotoxic potential. Cells were stimulated overnight with IL-12 and IL-18 (as above), then incubated with K562 target cells for the last 4 h in the presence of BFA and anti-CD107a-PE-Cy7 (BD Bioscience, Franklin Lakes, NJ, USA).
Data are expressed as the difference between the percentage of cytokine or CD107a-positive+ NK cells in the stimulated and unstimulated samples.
Data are expressed as the difference between the percentage of cytokine or CD107a-positive+ NK cells in the stimulated and unstimulated samples.
3
Cytokine Profile Analysis in CD4+ T Cells
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After 72 h of in vitro stimulation, the cells were incubated with a cell stimulation cocktail (1:500, eBioscience, USA). After 5 h of incubation, the cells were stained with anti-CD4-APC (BioLegend, USA) at room temperature for 30 min in the dark. After fixation and permeabilization, the cells were stained with anti-IFN-γ-PerCP-Cy5.5 (BioLegend, USA), anti-IL-2-PE (BioLegend, USA), and anti-TNF-α-FITC (BioLegend, USA) at room temperature for 30 min in the dark. Immunoglobulin IgG isotype-matched antibodies served as the negative controls. The cells were analyzed with the FACScan system.
4
Multiparameter Immune Cell Profiling
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Anti–CD3ε-PerCP (100326), anti–CD8a-AF700 (100730), anti–CD8a-APC (100712), anti–PD-1-PE/DZ594 (109116), anti–Ki67-FITC (652410), anti–TCF1-PE (655208), anti–IFN-γ-BV421 (505830), anti–IFN-γ-PerCp/Cy5.5 (505822), anti–TNF-α-APC/Cy7 (506344), anti–TIM-3-PE/Cy7 (119716), anti–CD45-BV510 (103138), anti–Ly108 (Slamf6)-PE (134606), anti–PD-1-FITC (135214), anti–CD45.1-PE (110708), anti–CD45.2-AF488 (109816), purified anti-mouse CD3ε (100340), and purified anti-mouse CD28 (102116) were obtained from Biolegend. Anti–CD8-FITC (D271-4) and tetramer-SIINFEKL-APC (TS-5001-2c) were purchased from MBL. Cell Stimulation Cocktail Plus Protein Transport Inhibitors and FOXP3/TF Staining Buffer Set were bought from eBioscience. Naive CD8a+ T Cell Isolation and Tumor Dissociation Kits were obtained from Miltenyi Biotec.
5
Multicolor Flow Cytometry Immunophenotyping
6
Comprehensive T Cell Cytokine Profiling
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Cryopreserved PBMCs from each participant at all time points were investigated simultaneously. After overnight rest of thawed PBMC, the cells were cultured in the presence of anti-CD107a-PE in complete RPMI containing 2 μg/mL of recombinant HBsAg (MyBioSource, USA) and 5 μg/mL of PHA (Sigma-Aldrich, USA) as the positive stimulation control or complete media alone as the unstimulated control at 37°C in a 5% CO2 incubator for 16–18 hr.
Cytokine-producing T cells analysis was performed as described previously [34 (link)]. Briefly, the overnight culture was further incubated with 5 μg/mL of brefeldin A and 1 μM of monensin (Sigma-Aldrich, USA) for 4 hr. Then, the cells were stained with anti-CD8-PE Alexa Fluor 610 (Life Technologies, USA), and anti-CD4-APC/Cy7 and anti-CD45RO-Pacific Blue (BioLegend, USA). After fixation and permeabilization, intracellular staining was performed by incubating the cells with anti-CD3-Krome Orange (Beckman Coulter, USA), and with anti-TNF-α-FITC, anti-IFN-γ-PerCP/Cy5.5, anti-IL-2-PE/Cy7, and anti-IL-10-APC (BioLegend, USA). At least 100,000 lymphocytes were collected for each sample by using Cyan ADP 9-color flow cytometer (Beckman Coulter, USA). Flow cytometric analysis of cytokine-producing or degranulation maker CD107a-expressing T cells was performed by using Kaluza software (Beckman Coulter, USA).
Cytokine-producing T cells analysis was performed as described previously [34 (link)]. Briefly, the overnight culture was further incubated with 5 μg/mL of brefeldin A and 1 μM of monensin (Sigma-Aldrich, USA) for 4 hr. Then, the cells were stained with anti-CD8-PE Alexa Fluor 610 (Life Technologies, USA), and anti-CD4-APC/Cy7 and anti-CD45RO-Pacific Blue (BioLegend, USA). After fixation and permeabilization, intracellular staining was performed by incubating the cells with anti-CD3-Krome Orange (Beckman Coulter, USA), and with anti-TNF-α-FITC, anti-IFN-γ-PerCP/Cy5.5, anti-IL-2-PE/Cy7, and anti-IL-10-APC (BioLegend, USA). At least 100,000 lymphocytes were collected for each sample by using Cyan ADP 9-color flow cytometer (Beckman Coulter, USA). Flow cytometric analysis of cytokine-producing or degranulation maker CD107a-expressing T cells was performed by using Kaluza software (Beckman Coulter, USA).
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