Antigen-specific responses were measured in peptide-stimulated thawed PBMC after overnight resting; 7·5 × 105 cells/well were stimulated in sterile conditions with peptide pools; ESAT-6/CFP-10 (E6C10) or Ag85, SEB or serum-free medium (AIM V; Gibco Invitrogen, Carlsbad, CA, USA) with 0·1% highly purified human albumin in short-term (6 h) cultures. Only PBMC with viability >85% were included. Brefeldin A (BD Bioscience) (final concentration 5 μg/ml) and monensin (BD Bioscience) (final concentration 5 μg/ml) were added at the time of stimulation with anti-CD107a 18 (link),19 . After 8 h rest at 4°C, cells were washed and stained with live/dead discriminator in azide-free and serum/protein-free phosphate-buffered saline (PBS) followed by CD4 and CD3 staining. PBMC were then washed, permeabilized (Cytofix-Cytoperm kit; BD Bioscience) according to the manufacturer's instructions and stained for intracellular cytokines (IFN-γ, TNF-α and IL-2). The following directly conjugated monoclonal antibodies were used: anti-CD3-peridinin chlorophyll (PerCP)-cyanin 5·5 (Cy5·5), anti-CD4-V500, anti-TNF-α-phycoerythrin (PE), anti-IFN-γ-PE-Cy7, anti-IL-2-allophycocyanin (APC), anti-CD107a-fluorescein isothiocyanate (FITC) (BD Bioscience) and live/dead discriminator Fixable Viability Dye eFluor® 450 (eBioscience, San Diego, CA, USA).
Anti ifn γ pe cy7
Manufactured by BD
Sourced in United States
Anti-IFN-γ PE-Cy7 is a fluorescent-conjugated antibody that binds to the interferon-gamma (IFN-γ) protein. It is used for the detection and quantification of IFN-γ-producing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm kit, by BD (5 mentions)
Monensin, by BD (3 mentions)
Anti cd3 fitc, by BioLegend (3 mentions)
Anti perforin pe, by BioLegend (3 mentions)
Anti cd8 apc, by BD (3 mentions)
Cd8 apc, by BD (3 mentions)
Anti cd4 fitc, by BD (3 mentions)
Brefeldin a, by BD (2 mentions)
Facsverse system, by BD (2 mentions)
Anti il 17a pe, by BioLegend (2 mentions)
Annexin 5 fitc and 7 aad, by BD (2 mentions)
Anti tnf α pe, by BD (2 mentions)
Anti cd4 pe cy7, by BioLegend (2 mentions)
Anti cd161 apc, by BioLegend (2 mentions)
Anti tcr vα7.2 brilliant violet 421, by BioLegend (2 mentions)
Anti cd8 perp cy5, by BioLegend (2 mentions)
Anti ccr6 pe cy7, by BioLegend (2 mentions)
Anti granzyme b pe cy7, by BioLegend (2 mentions)
Anti cxcr6 pe, by BioLegend (2 mentions)
Anti cd69 pe cy7, by BD (2 mentions)
25 protocols using anti ifn γ pe cy7
1
Multifunctional T Cell Responses Profiling
2
Comprehensive T Cell Phenotyping in Lungs and Spleens
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The analysis of CD4+ and CD8+ T cell responses in lung tissues and spleens by flow cytometry was performed as follows. Spleen and lung single-cell suspensions were stimulated with the RBD peptide pool for 5 h in the presence of 12.5 µg/mL Brefeldin A (BD Biosciences, East Rutherford, NJ, USA). Cells were then incubated on ice with a combination of fluorescent dye-labelled antibodies including anti-CD4-V500 (clone GK1.5; 1:200 dilution), anti-CD8α-APC-Fire750 (clone 53-6.7; 1:200 dilution), anti-CD11a/CD18-Pacific Blue (H155-78; 1:200 dilution), anti-CD103-PE (M290; 1:200 dilution), anti-CD69-FITC (H1-2F3; 1:200 dilution), anti-Ly6G-PerCP-Cy5.5 (clone 1A8; 1:200 dilution), anti-CD64-PerCP-Cy5.5 (clone X54-5/7.1; 1:200 dilution), anti-B220/CD45R-PerCP (clone RA3-6B2; 1:200 dilution), and Red LIVE/DEAD (1:1000 dilution). Following surface staining, cells were permeabilized using BD Biosciences Cytofix/Cytoperm kit (East Rutherford, NJ, USA) and stained with anti-IFN-γ-PE-Cy7 (XMG1.2; 1:200 dilution) and anti-TNF-α-APC (MP6-XT22; 1:200 dilution). Following incubation with the antibodies, cells were washed and resuspended before analysis on a BD FACSCanto II within 12 h.
3
Multiparametric Characterization of MAIT Cells
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Flow cytometry was performed as described using the following antibodies. The anti-CD3-FITC, anti-CD161-APC, and anti-TCR Vα7.2-Brilliant Violet 421(Biolegend, USA) were used to identify MAIT cells. Anti-CD4-PE/Cy7, anti-CD8-Perp/Cy5.5, anti-CCR6-PE/Cy7, anti-CXCR6-PE, anti-IL-17A-PE, anti-Granzyme B-PE/Cy7, and anti-Perforin-PE were obtained from Biolegend (USA). Anti-CD69-PE/Cy7, anti-PD-1-PE, anti-IFN-γ-PE/Cy7, anti-TNF-α-PE were obtained from BD (USA). Appropriate isotype control antibodies were used for each staining combination. We used 7-aminoactinomycin D(7-AAD) (BD Biosciences) staining to identify dead cells. Human MR1 tetramers loaded with a potent MAIT cell ligand-5-OP-RU or 6-FP were gifted from professor Li Bai. Apoptosis was allowed to progress and two channels were used to detect annexin V- FITC and 7-AAD (BD Biosciences) to determine the proportions of apoptotic cells. Data acquisition was achieved on BD FACS Verse system (BD Biosciences), and results were analyzed using FlowJo7.6 analysis software.
4
PBMC Isolation and Functional Assay
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PBMC were isolated from fresh blood using Ficoll and counted using Luna-FL cell counter (Logos Biosystems). Frozen PBMCs were thawed, counted and 1 × 106 PBMCs were stimulated with MVA-Venus (MOI = 1) for 24 h in a total volume of 200 µl of RPMI media with 5% of human serum in a 96 wells plate. Unstimulated cells and PHA (20 µg/ml) were used as negative and positive controls, respectively. Anti-CD107a-APC, Golgi Plug (Brefeldin A) and Golgi Stop (Monesin) were added 4 h before staining. Cells were washed with PBS and stained 15 min at room temperature with Live and dead near IR, anti-CD3-FITC (Biolegend 300440, 1:40), anti-CD4-BV421 (BD 566703, 1:50), anti-CD8-BV605 (Biolegend 301040, 1:100), anti-CD69-PE (BD 555531, 1:10), anti-CCR7-PE/Dazzle 594 (Biolegend 353236, 1:20) and anti-CD45RA-PerCP (Biolegend 304062, 1:20). After washing, cells were permeabilized with Fix/Perm (BD) for 20 min at 4 °C. Cells were washed with Perm/Wash (BD) and intracellular stained with anti-IFNγ-PECy7 for 30 minutes at 4 °C, washed and resuspended in FACS buffer until acquisition. Cells were acquired in Attune NxT cytometer (Thermofisher) and data was analyzed with Flow Jo X software following the gating strategy described in Supplementary Fig. 2 . Only two out of five DL3 volunteers had available PBMC samples for the analysis.
5
Comprehensive Leukocyte Immunophenotyping Protocol
6
Comprehensive Cytokine Profiling of HCV-Specific T Cells
7
Immunophenotyping of Cytotoxic T Cells
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The following antibodies were purchased from BD Pharmingen (San Jose, CA, USA): anti-CD44 APC-Cy7, anti-IFNγ PE-Cy7, CD8 PE, and CD8 APC, relevant isotype antibodies. Anti-CD62L PE and anti-IFNγ capture antibody (Clone R4-6A2) were purchased from eBioscience (San Diego, CA, USA). Anti-GFP FITC was purchased from Abcam (Cambridge, UK). SR-FLIVO-red was courtesy of ImmunoChemistry laboratories (Bloomington, MN, USA). All cell culture media was purchased from Invitrogen (Carlsbad, CA, USA). Recombinant IFNγ was purchased from R&D Systems (Minneapolis, MN, USA). Z-DVED-FMK, Ovalbumin (OVA) and SIINFEKL peptide were purchased from Sigma-Aldrich (St. Louis, MO, USA).
8
Cytokine Profile of Antigen-Specific T Cells
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1.0 × 106 PBMCs were stimulated with 1 μg/ml mesothelin peptide pool (Peptides & Elephants, Potsdam, Germany), medium control or PMA/Ionomycin (positive control, Sigma-Aldrich, St Louis, MI; USA) in R10 medium in the presence of Brefeldin A (10μg/mL, Sigma-Aldrich St Louis, MI, USA) for 6 hours at 37°C. Stimulation was stopped by transferring the cells to a 4°C refrigerator, followed by washing with FACS buffer and staining with the following reagents: anti-CD3 Pacific blue (BD Biosciences, CA, USA), anti-CD4 PerCP-Cy5.5 (BD Biosciences, CA, USA) and anti-CD8 APC-Cy7 (BD Biosciences, CA, USA). After a 15-minute incubation at 4°C, the cells were washed and fixed with a Fix/Perm reagent (Beckman coulter, CA, USA), followed by a further 30-minute incubation at 4°C with an intracellular antibody mix (anti-TNFα APC (BD Biosciences CA, USA), anti-IFN-γ PE-Cy7 (BD Biosciences CA, USA), anti-IL-2 PE (BD biosciences CA, USA) and anti-IL-17 FITC (BioLegend, CA, USA)). The stained cells were then washed with FACS buffer and acquired on a FACSAria flow cytometer (BD Biosciences, Stockholm, Sweden). Data was analysed using FlowJo software (Treestar Inc.).
9
Immunophenotyping of Granulocytic Myeloid-Derived Suppressor Cells
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All antibodies were purchased from Biolegend (San Diego, CA, USA), except for anti‐CD14‐APCH7, anti‐CD3‐PerCPCy5.5, anti‐CD107a‐FITC and anti‐IFN‐γ‐PECy7, which were purchased from BD Biosciences (Franklin Lakes, NJ, USA). To determine the frequencies of gMDSCs, peripheral blood mononuclear cells were first stained with live/dead‐BV510 (Life Technologies, Waltham, MA, USA) and surface antibodies and then incubated for 20 minutes at 4°C in the dark. gMDSCs were counted as CD15+HLA−DR−CD14− cells derived from CD33+CD11b+ myeloid cells. For intracellular staining, cells were permeabilized with the Cytofix/Cytoperm Solution Kit (BD Bioscience) and stained with anti‐arginase I‐FITC (R&D, Minneapolis, MN, USA) or anti‐IFN‐γ‐PECy7; a matched isotype antibody was used as a negative control. After staining, cells were fixed in 1% paraformaldehyde and analysed using a FACS Verse flow cytometer and FlowJo software. For morphologic analysis, flow cytometric‐isolated gMDSCs were stained with a Wright‐Giemsa kit (Solarbio, Beijing, China) following the manufacturer's instructions.
10
Intracellular Cytokine Staining Protocol
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Intracellular staining was done as previously described (Lindenstrøm et al., 2013 (link)). Briefly, 1–2 × 106 cells were stimulated in vitro in V-bottom 96-well plates at 37 °C in RPM1 with 10% FBS and 10 μg/ml Brefeldin A. The cells were stimulated with 2 μg/ml ESAT-61-15 peptide, 1 μg/ml anti-CD28 (clone 37.51) or anti-CD49d (clone 9C10-MFR4.B) for 5–6 h. Cells were washed with FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) followed by staining with surface antibodies as necessary. Fixation and permeabilization was carried out using the Cytofix/Cytoperm kit (BD Biosciences) as per manufacturer's instructions followed by intracellular staining with fluorochrome conjugated antibodies against the respective cytokines. Some of the cytokines antibodies used were anti–IFN-γ PE-Cy7 (BD Biosciences Cat# 557649 RRID:AB_396766 ), anti–TNF–PE (BD Biosciences Cat# 554419 RRID:AB_395380 ), IL-2–APC–Cy7 (BD Biosciences Cat# 560547 RRID:AB_1727544 ) and IL-17A–PerCP–Cy5.5 (Thermo Fisher Scientific Cat# 45–7177-82 RRID:AB_925753 ).
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