IFN-γ production by human T cells was assessed by intracellular staining as previously described [17 ]. Briefly, PBMCs were seeded in RPMI 1640 supplemented with 10% FCS in the presence of brefeldin A (1:1000; #555,029; BD Bioscience) and were either left unstimulated or stimulated for 6 h in the presence of CD2/CD3/CD28 beads (1:40; #10,970, Stem Cell technologies). Cells were then harvested and stained for CD3-PE (1:50; # 12–0038-42, Thermo Fisher Scientific) in the presence of a viability marker (#L34957, Thermo Fisher Scientific), followed by fixation and permeabilization (#88–8824-00, Thermo Fisher Scientific). Cells were stained for intracellular IFN-γ-AF700 (1:100; #561,024, BD) and TNF-APC (1:50; # 130–117-531, Miltenyi) and analyzed on a Fortessa X-20 Cell Analyzer (BD).
Ifn γ af700
Manufactured by BD
Sourced in United States
IFN-γ-AF700 is a fluorescently labeled antibody that targets interferon-gamma (IFN-γ). It is designed for use in flow cytometry applications to detect and quantify IFN-γ-producing cells.
Automatically generated - may contain errors
Lab products found in correlation
Il 2 pe, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Fortessa x 20 cell analyzer, by BD (1 mentions)
Cd3 pe, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by BD (1 mentions)
Annexinv pacific blue, by Thermo Fisher Scientific (1 mentions)
Cd19 apc h7, by BD (1 mentions)
Cd3 ecd, by Beckman Coulter (1 mentions)
Cd8 apc, by BD (1 mentions)
Tnf α fitc, by BD (1 mentions)
T bet bv421, by BD (1 mentions)
Ki67 fitc, by BD (1 mentions)
Tigit pe cy7, by BD (1 mentions)
Granzyme b af700, by BD (1 mentions)
Anti cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Cd45ra ecd, by Beckman Coulter (1 mentions)
5 protocols using ifn γ af700
1
IFN-γ and TNF Production in T Cells
2
Multiparametric Cell Immunophenotyping
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Aqua Viability Dye (AqVi; Invitrogen) exclusion was used to identify viable cells. The antibodies used for these studies were: CD3-ECD (Beckman Coulter), CD8-APC (BD Pharmingen), CD19-APCH7 (BD Biosciences), HIV-1 p24-PE (Beckman Coulter), IL-17-V450 (BD biosciences), IFN-γ-AF700 (BD biosciences) and AnnexinV-Pacific Blue (Life Technologies). Isotype controls were used to establish the gates for IL-17 (Mouse IgG1- V450, BD Biosciences) and IFNγ (Mouse IgG1-AF700, BD biosciences).
3
Multiparametric Flow Cytometry of Immune Markers
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Peripheral blood mononuclear cells were cultured in RPMI‐1640 medium (GIBCO, Grand Island, NY, USA) containing 10% FBS and stimulated with anti‐CD3/CD28 (2 μg/mL and 5 μg/mL; Ebioscience) or phorbol myristate acetate (PMA)/ionomycin (50 ng/mL and 1 μg/mL), plus Golgiplug (BD Biosciences) for 5 h. The cells were then surface‐stained with CD3‐BV786, CD4‐APC‐H7, CD8‐BV421, PD‐1‐BV711, or TIGIT‐PE‐Cy7, and intracellularly stained with IFN‐γ‐AF700, TNF‐α‐FITC, or IL‐2‐PE (BD Biosciences) antibodies. For ki67, perforin, T‐bet, or Eomes staining, PBMCs were surface‐stained with CD3‐BV786, CD4‐ APC‐H7, CD8‐BV650, PD‐1‐BV711, or TIGIT‐PE‐Cy7, and intracellularly stained with perforin‐APC, ki67‐FITC, Granzyme B‐AF700, T‐bet BV421, or Eomes PE (BD Biosciences) antibodies. A Fixable Viability Dye eFluor® 506 (Ebioscience) was used to assess cell viability.
4
Functional Assays for NK Cell Activation
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For functional experiments, PBMCs were thawed, rested overnight, and then mixed with K562 cells or RAJI cells or P815 cells at a ratio of 10:1 in U-bottomed 96-well plates and incubated for 6 h at 37°C and 5% CO2. For redirected ADCC assays, P815 cells were incubated with 5 μg/mL of the indicated anti-NKG2C. For ADCC experiments, RAJI cells were incubated with 1 μg/mL of rituximab (anti-CD20). Preceding the assay, CD107a-FITC (H4A3) was added together with brefeldin (Golgi Plug, 1/1000, Becton Dickinson) and monensin (Golgi Stop, 1/1500, Becton Dickinson). For polyfunctional assays, cells were permeabilized (Fixation & Permeabilization Buffers, eBioscience) and then stained with intracellular IFNγ-AF700 (Becton Dickinson) and TNFα-eF450 (eBioscience) and analyzed by flow cytometry. Pie charts were generated using the Spice software (26 (link)).
5
Multiparameter Flow Cytometry Analysis
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The following monoclonal antibodies (mAbs) were used in different combinations. CD8-PB, CD3-APC-H7, PD-1-PECy7, PD-1-PB, IFN-γ-AF700, IL-2-PE, CD4-PB, granzyme B-AF700, perforin-APC were purchased from Becton Dickinson (BD, San Diego, CA), CD45RA-ECD, CD4-ECD from Beckman Coulter (Fullerton, CA, USA), CCR7-FITC from R&D Systems (Minneapolis, MN, USA), 2B4-PECY5.5, CD160-APC, CD160-PE from BioLegend (San Diego, CA, USA), CD4-eFluor650NC, CD8-eFluor625NC from eBioscience.
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