Intracellular cytokine staining (ICS) was performed as described.16 (link) Briefly, T cells were cocultured with SARS-CoV-2 strains Victoria 01/20, Delta or Omicron-infected BCLs-ACE2 at an E:T ratio of 1:2 for 6 h together with GolgiPlug and GolgiStop. The cells were then surface stained with PE-anti-CD107a (BD Biosciences, 1:20). Dead cells were labelled using Live/Dead Fixable Aqua dye (Invitrogen, 1:1000). After fixated with Cytofix/Cytoperm (BD Biosciences), CD8+ T cells were stained with BV421-anti-CD8 (Biolegend, 1:33), PE-Cy7-anti-IFNγ (BD Biosciences, 1:33), APC-anti-TNFα (eBioscience, 1:500) and APC-H7-anti-MIP1β (BD Biosciences, 1:33); CD4+ T cells were stained with APC-anti-CD4 (Thermofisher, 1:33), PE-Cy7-anti-IFNγ (BD Biosciences, 1:33), APC-H7-anti-TNFα (Biolegend, 1:33) and BV421-anti-IL2 (Biolegend, 1:33). Negative controls without virus infection were run for each sample. All samples were acquired on Attune NxT Flow Cytometer (software v.3.2.1) and analyzed using FlowJo v.10 software (FlowJo LLC).
Pe cy7 anti ifnγ
Manufactured by BD
Sourced in United States
PE-Cy7-anti-IFNγ is a fluorescently-labeled antibody used for the detection and quantification of interferon-gamma (IFNγ) protein in flow cytometry applications. The antibody is conjugated to the PE-Cy7 fluorescent dye, which allows for multicolor flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (4 mentions)
Saponin, by Merck Group (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Cytofix cytoperm, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Pacblue anti cd4, by BD (2 mentions)
Anti il 10 pe, by Thermo Fisher Scientific (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Anti cd107a pe, by BD (1 mentions)
Bv421 anti cd8, by BioLegend (1 mentions)
Anti cd4 apc, by Thermo Fisher Scientific (1 mentions)
Live dead fixable aqua dye, by Thermo Fisher Scientific (1 mentions)
Apc anti tnfα, by Thermo Fisher Scientific (1 mentions)
Attune nxt flow cytometer, by FlowJo (1 mentions)
Anti perforin pe, by BioLegend (1 mentions)
Anti cd4 bv605, by BioLegend (1 mentions)
Anti cd3 bv421, by BioLegend (1 mentions)
96 well cell culture plate, by Corning (1 mentions)
Anti tnf α apc, by BD (1 mentions)
9 protocols using pe cy7 anti ifnγ
1
Intracellular Cytokine Staining of SARS-CoV-2-Specific T Cells
2
Cytokine and Granule Profiling of Tumor-specific CD4+ T Cells
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Tumor-specific CD4 T clones were stimulated with serial dilutions of the specific peptides or left untreated for 6 hours at 37°C in the presence of brefeldin A (10 μg/ml; Sigma-Aldrich). Cells were then stained in PBS, 0.2% BSA, 5 mM EDTA, and 0.2% NaN3 with BV421 anti-CD3 (BioLegend) and BV605 anti-CD4 (BioLegend) at 4°C for 30 min, fixed in PBS 1% formaldehyde, 2% glucose, and 5 mM NaN3 for 20 min at room temperature, and, lastly, stained in PBS, 0.2% BSA, 5 mM EDTA, 0.2% NaN3, and 0.1% saponin (Sigma-Aldrich) with either (i) PE-Cy7 anti-IFNγ (BD Biosciences), PerCP-Cy5.5 anti-TNFα (BioLegend), APC anti–IL-13 (BioLegend), and A700 anti–IL-17a (BioLegend) or (ii) PE anti-perforin (BioLegend), A700 anti-granzyme A (BioLegend), ECD anti-granzyme B (BD Biosciences), PerCP710 anti-granzyme K (eBioscience), APCeF660 anti-granzyme M (eBioscience), and A488 anti-granulysin (eBioscience) for 30 min at 4°C before acquisition on an LSR II SORP (Beckman Coulter) flow cytometer. Percentages of granule/cytokine-positive T cells were analyzed using FlowJo software (v.10.4.2). EC50 values were derived by dose-response curve analysis [log(agonist) versus response] using Prism software. Noncytokine clones, indicated in the added gray boxes, for which an EC50 value could not be determined accurately, were not included in the statistical analyses.
3
PBMC Cytokine Response to Mycobacterial Antigens
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Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Stemcell, Canada). PBMCs (5 × 105 cells/well) were seeded in 96-well cell culture plate (Corning, NY, USA) and stimulated for 4 days with 15 µg/ml of Rv1738, Rv2659c or ESAT-6 at 37 °C with 5% CO2. Phorbol 12-myristate 13-acetate (Sigma, USA) at 25 ng/ml and Ionomycin (Sigma, USA) at 1 µg/ml were used as positive controls. Non-stimulated cells were used as negative controls. Cells were incubated in the presence of 10 µg/ml Brefeldin A (Sigma, USA) for 4 h before the end of culture. The PBMCs were then harvested and stained with the following cell surface markers: Alexa fluor700 anti-CD3, PerCP-Cy5.5 anti-CD4, APC-Cy7 anti-CD8 (at 4 °C for 15 min). Subsequently, cells were fixed and permeabilized with a Cytofix/Cytoperm Kit (BD Biosciences, USA) and then stained with PE-Cy7 anti-IFN-γ, FITC anti-IL-2 and APC anti-TNF-α at 4 °C for 30 min. All antibodies were purchased from BioLegend, USA. Data were acquired using a FACS Canto II (BD Biosciences, USA) and were analysed by FlowJo software (Tree Star, USA).
4
Multiparameter Flow Cytometry Analysis
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In vitro polarized or ex vivo–derived T cells were restimulated with PMA/ionomcyin for 5 h. Brefeldin A was added after 1 h of polyclonal restimulation. Cells were hereafter treated with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Invitrogen). Then cells were fixed with 2% paraformaldehyde (PFA; Sigma-Aldrich) for 20 min at room temperature. For FACS analysis, cells were stained in 0.5% saponin (Sigma-Aldrich). The following antibodies were used: PacBlue–anti-CD4, PE-Cy7–anti–IFN-γ (both BD), and PE–anti–IL-10 (eBioscience). Foxp3 co-staining was done subsequently to the intracellular cytokine staining using the eBioscience Foxp3 Transcription Factor Staining Buffer Set and according to manufacturer’s instructions. FACS analysis was performed on a FACS-Canto (BD).
5
Comprehensive Immunophenotyping of Gamma-Delta T Cells
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Antibodies for flow cytometry analysis: BV310 anti-CD3, FITC anti-TCRVγ9Vδ2, PE or PeCy5 anti-CD107a, PeCy7 anti-IFNγ, PE anti-TIM3, PE anti-Galectin9, PeCy7 anti-PD1, APC anti-PDL1, PeCy5 anti-CD80, PE anti-CD80, PeCy5 anti-HLAABC, AF647 anti-CD31, PeCy7 anti-CD38, FITC anti-CD226, FITC anti-CD112, FITC anti-CD155, PE anti-LFA1, and isotype controls (BD Biosciences, Pont de Claix, France); BV421 anti-CD69 and isotype control (Miltenyi Biotech, Paris, France); PE anti-HLAE (eBiosciences); PE anti-ULPB2,5,6 (R&D Systems, Minneapolis, USA); APC anti-MICA/B (Biolegend, St-Quentin-en-Yvelines, France); PE anti-ICAM1 and PE anti-ICAM3 (Immunotech, Marseille, France); PE anti-LFA3 (Beckman Coulter, Fullerton, CA, USA).
Blocking antibodies: anti-BTN3A1 1 h at 10 μg/mL (103.2 clone, kindly gifted by ImCheck Therapeutics, Marseille, France), anti-γδTCR 1 h at 0.5 mg/mL (B1 clone, Biolegend), anti-ICAM1 (W-CAM-1 clone, Thermo fisher, Villebon sur Yvette, France) and anti-CD31 1 h at 10 μg/mL (HEC7 clone, Thermo fisher, Villebon sur Yvette, France). The exoenzyme C3 transferase was used as RHO inhibitor I overnight at 2 μg/mL (Cytoskeleton, Inc. Denver, USA).
Blocking antibodies: anti-BTN3A1 1 h at 10 μg/mL (103.2 clone, kindly gifted by ImCheck Therapeutics, Marseille, France), anti-γδTCR 1 h at 0.5 mg/mL (B1 clone, Biolegend), anti-ICAM1 (W-CAM-1 clone, Thermo fisher, Villebon sur Yvette, France) and anti-CD31 1 h at 10 μg/mL (HEC7 clone, Thermo fisher, Villebon sur Yvette, France). The exoenzyme C3 transferase was used as RHO inhibitor I overnight at 2 μg/mL (Cytoskeleton, Inc. Denver, USA).
6
Multiparameter flow cytometry analysis
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Single-cell suspensions were incubated with a CD16/32 (clone 2.4G2) antibody (BioLegend, catalog no. 101301, 1:200) to block nonspecific staining on ice for 15 minutes, and the LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (Thermo Fisher Scientific, catalog no. L34960) was used to exclude dead cells for further analyses. Then surface staining was performed with 2 μg/mL of the following fluorochrome-conjugated antibodies at 4°C for 30 minutes: PerCP/Cy5.5 anti-CD45 (30-F11, catalog no. 103131), PE/Cy7 anti-CD11c (N418, catalog no. 117317), FITC anti-CD8a (53-7.6, catalog no. 100705), APC/Cy7 anti-CD4 (GK1.5, catalog no. 100413), PerCP/Cy5.5 anti-CD11b (M1/70, catalog no. 101227), APC anti-F4/80 (BM8, catalog no. 123115), and APC anti-CXCR3 (CXCR3-173, catalog no. 126511) from BioLegend. For detecting IFNγ+ cells, phorbol 12-myristate 13-acetate (Beyotime, catalog no. S1819) and Ionomycin (Beyotime, catalog no. S1672) were used to stimulated cells. After stimulation, cells were fixed and permeabilizated with fixation and permeabilization solutions kit (BD, catalog no. 554714), and stained with PE/Cy7 anti-IFNγ (XMG1.2, catalog no. 505825) at 4°C for 30 minutes. All the samples were acquired with Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed with FlowJo software (Tree Star, Inc.).
7
Flow Cytometry Analysis of T Cell Subsets
8
Intracellular Cytokine Analysis of T Cells
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The intracellular production of IFNγ and of IL17A by T lymphocytes from PBMC and tissue infiltrates was analyzed as follows: the cells (resuspended in culture medium conditioned with 10% autologous plasma at a concentration of 1 × 107/ml) were stimulated with phorbol-12-myristate-13-acetate (PMA 50 ng/ml, Sigma) and ionomycin (2 μg/ml, Sigma) for 5 hours at 37°C. Brefeldin A (BFA 10 μg/ml, Sigma) was added to the cells for the last 4 hours of incubation. After washings, the samples were stained with fluorochrome-conjugated mAbs specific for surface markers [PerCPCy5.5 anti-CD3 (BD), APCCy7 anti-CD8 (e-Biosciences) and Violet Live/Dead Fixable Dead Cell stain (Life Technologies, CA, USA)], before fixing and permeabilizing the lymphocytes with the Cytofix/Cytoperm kit (BD Bioscience) following the manufacturer's instructions. The cells were washed in Perm-Wash buffer (BD Bioscience) and incubated with Pe-Cy7 anti-IFNγ (BD) or FITC anti-IL17A (e-Biosciences) mAbs. Thereafter, the samples were washed in Perm-Wash buffer, fixed with FACS Lysing solution (BD Bioscience) and stored at 4°C. The cytokine profile was analyzed using a FACS Canto II flow cytometer (BD Bioscience) by the FACS DIVA software. The gating strategy for phenotypic analyses is shown in Supplementary Figure 3 .
9
Multiparametric Flow Cytometry for Immune Cell Profiling
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Surface staining was performed using following antibodies: FITC-anti-CD4 (Clone RM4-5; 1:200), PerCP-anti-CD8 (Clone 53-6.7; 1:100), PacBlue-anti-B220 (Clone RA3-6B2, 1:200), APC-Cy7-anti-CD25 (Clone PC61, 1:200), PE-anti-CD44 (Clone IM7, 1:200), and APC-anti-CD86 (Clone B7-2, 1:200) (all from BD Biosciences, San Jose, CA, USA).
For intracellular staining, cells were restimulated for 5 h with 10 ng/ml phorbol myristate acetate and 1 µg/ml ionomycin, in a tissue culture incubator at 37°C (both Sigma-Aldrich). Ten micrograms per milliliter brefeldin A (Sigma-Aldrich) were added to the cell suspensions after 1 h of polyclonal restimulation. Then cells were treated with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies) and hereafter fixed with 2% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Cells were stained in 0.5% saponin (Sigma-Aldrich) using following antibodies: PacBlue-anti-CD4 (Clone RM4-5; 1:400), PE-Cy7-anti-IFN-γ (Clone XMG 1.2; 1:400) (both from BD Biosciences), FITC-anti-IL17A (Clone TC11-18H10.1; 1:200, BioLegend, San Diego, CA, USA), PE-anti-IL10 (Clone JESS-16E3; 1:100), and APC-anti-IL22 (Clone IL22JOP; 1:100) (both from eBioscience). All data were acquired on a MACSQuant analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and were analyzed with FlowJo Software v10.1 (Tree star, Ashland, OR, USA).
For intracellular staining, cells were restimulated for 5 h with 10 ng/ml phorbol myristate acetate and 1 µg/ml ionomycin, in a tissue culture incubator at 37°C (both Sigma-Aldrich). Ten micrograms per milliliter brefeldin A (Sigma-Aldrich) were added to the cell suspensions after 1 h of polyclonal restimulation. Then cells were treated with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies) and hereafter fixed with 2% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Cells were stained in 0.5% saponin (Sigma-Aldrich) using following antibodies: PacBlue-anti-CD4 (Clone RM4-5; 1:400), PE-Cy7-anti-IFN-γ (Clone XMG 1.2; 1:400) (both from BD Biosciences), FITC-anti-IL17A (Clone TC11-18H10.1; 1:200, BioLegend, San Diego, CA, USA), PE-anti-IL10 (Clone JESS-16E3; 1:100), and APC-anti-IL22 (Clone IL22JOP; 1:100) (both from eBioscience). All data were acquired on a MACSQuant analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and were analyzed with FlowJo Software v10.1 (Tree star, Ashland, OR, USA).
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