Ifn γ

Manufactured by Beckman Coulter

Sourced in United States

IFN-γ is an in-vitro diagnostic reagent used for the quantitative determination of Interferon Gamma (IFN-γ) in human whole blood samples. It is designed for use with the Beckman Coulter QuantiFERON®-TB Gold Plus (QFT-Plus) test kit.

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Anti-IFN-γ-FITC (3 citations) PE-conjugated anti-IFN-γ (3 citations) PE-conjugated anti-IFN-γ (clone 45.15) (2 citations)

6 protocols using ifn γ

Multiparameter Flow Cytometry Analysis

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The following fluorochrome-conjugated monoclonal antibodies were used in this study: CD3, CD4, CD8, CD19, and IFNγ from Beckman Coulter (Indianapolis, IN); CD71, HLA-A2, and HLA-A, B, C from BioLegend (San Diego, CA); CD95 (Fas) and CD107a from BD Biosciences (San Jose, CA); and CD34 (QBEnd-10) from Abnova (Taipei, Taiwan). Cell viability was assessed using 7-amino actinomycin D (7-AAD) (BD Biosciences) staining. We used the Gallios Flow Cytometer (Beckman Coulter) to acquire flow cytometric data and Kaluza Analysis Software (Beckman Coulter) to analyze data and for graphical representation.

Quach D.H., Becerra-Dominguez L., Rouce R.H, & Rooney C.M. (2019). A strategy to protect off-the-shelf cell therapy products using virus-specific T-cells engineered to eliminate alloreactive T-cells. Journal of Translational Medicine, 17, 240.

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Peripheral Blood Mononuclear Cell Isolation and Cytokine Induction

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Approximately 5 mL of peripheral blood should be collected in heparin tubes (BD
Biosciences, San Jose, CA, USA) and placed on a rotary plate overnight at 10
rpm. Peripheral blood mononuclear cells (PBMC) can be isolated from whole blood
using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Uppsala, Sweden). To
induce cytokine production, cells should be stimulated with
paramethoxyamphetamine (50 ng/mL; Sigma-Aldrich, St Louis, MO, USA), ionomycin
(500 ng/mL; Sigma-Aldrich, St Louis, MO, USA), and GolgiStop (1:1000; BD
Biosciences, San Jose, CA, USA) for 5 hours. The following antibodies may be
used: Foxp3, CD11c (BD Bioscience, Franklin Lakes, NJ, USA), IFN-γ, CD8 (Beckman
Coulter, Mississauga, ON, Canada), CD3, CD4, CD45RA, CD62L, T cell receptor
(TCR) γ/δ, CD56, CD16, CD19, CD27, IgD, CD14, CD25, and IL-17 (Biolegend, San
Diego, CA, USA).

Fleischer T., Chang T.T., Chiang J.H, & Yen H.R. (2018). A Controlled Trial of Sheng-Yu-Tang for Post–Hematopoietic Stem Cell Transplantation Leukemia Patients: A Proposed Protocol and Insights From a Preliminary Pilot Study. Integrative Cancer Therapies, 17(3), 665-673.

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Cytotoxicity and Cytokine Profiling of NK Cells

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CD107a expression on NK cells and the intracellular cytokine staining (ICS)-based assay were used to measure cytotoxic activity (degranulation) and cytokine expression as described previously with some modifications (21 (link), 43 (link)). Briefly, mononuclear cells (PBMCs) isolated from fresh whole blood were incubated overnight at 37°C with 5% CO₂ at 1:1 ratio with the K562 cell line, which was used as target cells, in the presence of a CD107a monoclonal antibody, brefeldin A (5 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) and monensin (6 μg/mL; Sigma-Aldrich), which were added concomitantly to the cells. Spontaneous basal degranulation and cytokine production were measured by incubating PBMCs under the same conditions without target cells. After incubation, anti-CD3, anti-CD56, and anti-CD16 were added, followed by antibodies for the ICS-based assay (IFN-γ, TNF, TGF-β, and IL-10; Beckman-Coulter and BD Biosciences). These evaluations were assessed by multiparametric flow cytometry using the XL-MCL and Gallios equipment from Beckman-Coulter.

Giacoia-Gripp C.B., Cazote A.D., da Silva T.P., Sant'Anna F.M., Schmaltz C.A., Brum T.D., de Matos J.A., Silva J., Benjamin A., Pilotto J.H., Rolla V.C., Morgado M.G, & Scott-Algara D. (2019). Changes in the NK Cell Repertoire Related to Initiation of TB Treatment and Onset of Immune Reconstitution Inflammatory Syndrome in TB/HIV Co-infected Patients in Rio de Janeiro, Brazil—ANRS 12274. Frontiers in Immunology, 10, 1800.

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Characterization of NK Cell Phenotype

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NK cells were isolated as described above and incubated for 20 min at 4°C with antibodies against CD158a (Beckman Coulter, CA, USA), CD158b (Beckman Coulter), CD56 (Beckman Coulter), or CD16 (Beckman Coulter) or antibodies against LAG3 (Biolegend), TIGIT (Biolegend), CTLA4 (Biolegend), CD57 (Biolegend), NKG2A (Biolegend), CD107A (Biolegend), Nkp46 (Biolegend), or NKG2D (Biolegend). After membrane staining, the cells were fixed with Fixative Reagent (Beckman Coulter) at room temperature for 15 min, then permeabilized with 300 µl permeabilizing reagent (Beckman Coulter) to detect intracellular protein expression. Permeabilized cells were incubated for 15 min at room temperature with antibodies against Granzyme B (Biolegend), perforin (Beckman Coulter), or IFN‐γ (Beckman Coulter). Flow cytometry was conducted on a DxFlex system (Beckman Coulter), and data were analysed using FlowJo software (v10.5.3).

Li X., Chen M., Wan Y., Zhong L., Han X., Chen X., Xiao F., Liu J., Zhang Y., Zhu D., Xiang J., Liu J., Huang H, & Hou J. (2022). Single‐cell transcriptome profiling reveals the key role of ZNF683 in natural killer cell exhaustion in multiple myeloma. Clinical and Translational Medicine, 12(10), e1065.

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Comprehensive Immune Cell Profiling

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Antibodies: CD279 (PD1) (Clone PD1.3 Beckman Coulter), CD45RA (Clone ALB11 Beckman Coulter), CD185 (CXCR5) (Clone J252D4 Biolegend), CD4 (Clone 13B8.2 Beckman Coulter), CD20 (Clone B9E9 Beckman Coulter), CD19 (Clone J4.119 Beckman Coulter), CD38 (Clone HB-7 Biolegend), CD27 (Clone M-T271 Biolegend), IGD (Clone IA6-2 Biolegend), CD11c (Clone BU15 Beckman Coulter), CD14 (Clone RMO52 Beckman Coulter), CD16 (Clone 3G8 Beckman Coulter), CD24 (Clone ALB9 Beckman Coulter), CD45 (Clone J.33 Beckman Coulter), CD3 (Clone UCHT1 Beckman Coulter), CD8 (Clone B9.11 Beckman Coulter), CD56 (Clone N901 Beckman Coulter), CD57 (Clone NC1 Beckman Coulter), CD25 (Clone B1.49.9 Beckman Coulter), CD127 (Clone R34.34 Beckman Coulter), CD159a (NKG2A)(Clone S19004C Biolegend), CD337 (NKp30) (Clone P30-15 Biolegend), IFN-γ (Clone 45.15 Beckman Coulter), IL-21 (Clone 3A3-N2 Biolegend), IL-17 (Clone BL168 Beckman Coulter), granzyme (Clone QA16A02 Biolegend), perforin (Clone B-D48 Biolegend)
Flow Cytometer: Navios, Beckman Coulter
Cytometric Bead Array: RAISECARE; Analysis Software: Kaluza, LEGEND plex.

Jin X., Yan Z.H., Lu L., Lu S., Zhang G, & Lin W. (2021). Peripheral Immune Cells Exhaustion and Functional Impairment in Patients With Chronic Hepatitis B. Frontiers in Medicine, 8, 759292.

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Comprehensive Immune Cell Profiling

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The following antibodies were used: anti-human CD19, CD21, CD38, TBNK 6-Color Kit and isotype controls (BD Biosciences), CD24, CD1d, IL-10, FoxP3 (eBiosciences), CD3, CD4, CD5, CD10, CD22, CD25, CD45, IFN-γ, TNF-α, IL-4 and isotype controls (all from Beckman Coulter), IgM (DAKO), TGF-β1 and isotype controls (Biolegend). Cell staining and flow cytometry analysis were performed according to routine laboratory methods, as described in the Supplementary Information. Stained cells were analyzed via flow cytometry analysis using a FACS Canto II (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Wang W., Yuan X., Chen H., Xie G., Ma Y., Zheng Y., Zhou Y, & Shen L. (2015). CD19+CD24hiCD38hiBregs involved in downregulate helper T cells and upregulate regulatory T cells in gastric cancer. Oncotarget, 6(32), 33486-33499.

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