Ifnγ apc cy7

Manufactured by BioLegend

IFNγ-APC-Cy7 is a fluorescently conjugated antibody that specifically binds to the cytokine interferon-gamma (IFNγ). It is designed for use in flow cytometry applications to detect and quantify IFNγ-producing cells.

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IFNγ-APC/Cy7 Clone 4S.B3 APC-Cy7-conjugated anti-IFNγ (clone 4S.B3) APC-Cy7

7 protocols using ifnγ apc cy7

Characterizing iPSC-derived T Cells

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For detection of CD107a and IFNγ expression of iPSC-T cells, T cells were co-cultured with B-LCLs cells at a 1:1 ratio in a 96-well plate for 5 h in 100 μL of RPMI-1640 supplemented with glutamine, 1× Monensin (BioLegend), 10% FBS, and anti-CD107a-APC antibody (BioLegend, Clone H4A3, 1 μL). Then the cell surface was stained with anti-CD20-PE-Cy7 (BD Pharmingen) and anti-EGFR-PE (BioLegend). Samples were washed and fixed with Fixation Buffer (BioLegend) for 20 min. Samples were washed twice with Permeabilization Wash Buffer (BioLegend) and were stained with IFNγ-APC-Cy7 (BioLegend) for 20 min. The washed and resuspended cells were analyzed using BD FACSAria II.

Takayanagi S.I., Wang B., Hasegawa S., Nishikawa S., Fukumoto K., Nakano K., Chuganji S., Kato Y., Kamibayashi S., Minagawa A., Kunisato A., Nozawa H, & Kaneko S. (2023). Mini-TCRs: Truncated T cell receptors to generate T cells from induced pluripotent stem cells. Molecular Therapy. Methods & Clinical Development, 31, 101109.

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Multicolor Flow Cytometry Analysis of T Cell Cytokines

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Single-cell preparations of spleen were obtained as previously described (47 (link)). Splenocytes were stimulated with each type of the CPSs or with the LPS of χ3761 (50 ng/2 × 10⁶ cells) in the presence of Protein Transport Inhibitor Mixture (eBioscience). The cells were then incubated for 8 h at 37 °C with 5% CO₂. Before staining, dead cells were excluded using Zombie Red (Biolegend) and mouse FcR were blocked with the rat anti-mouse CD16/CD32 monoclonal antibody (eBioscience). For surface staining, cells were incubated with fluorophore-conjugated antibodies on ice for 30 min. The eBioscience Intracellular Fixation and Permeabilization Buffer Set was used for intracellular staining. Cells were run on a BD LSRFortessa cell analyzer and analyzed with FlowJo software (TreeStar).
For detection of cytokine expression in CD4⁺ T cells at 21 d after primary immunization, the following antibodies purchased from Biolegend were used, including rat anti-mouse CD3-Alexa Fluor 700, CD4-FITC, IFN-γ–APC/Cy7, APC/Cy7-conjugated rat IgG1κ (isotype), IL-4–Alexa Fluor 647, Alexa Fluor 647-conjugated rat IgG1κ (isotype). Data are presented as described in the figure legends.

Su H., Liu Q., Bian X., Wang S., Curtiss R I.I.I, & Kong Q. (2020). Synthesis and delivery of Streptococcus pneumoniae capsular polysaccharides by recombinant attenuated Salmonella vaccines. Proceedings of the National Academy of Sciences of the United States of America, 118(2), e2013350118.

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Splenocyte Isolation and Characterization

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Spleens were collected and mashed in 70-μm cell strainers with complete media. Red blood cells were lysed with RBC lysis buffer (eBioscience). For surface staining, cells were blocked with anti-mouse CD16/32 (eBioscience), stained with fluorochrome-conjugated antibodies, and analyzed with BD FACSAria II flow cytometer (BD Biosciences, San Jose, CA). For intracellular staining, Foxp3 Fixation/Permeabilization kit (eBioscience) was used. Anti-mouse antibodies used in this study include the following: CD3-APC, CD4-PE-Cy7, CD8-FITC, IL-10-PerCP-Cy5.5, IFNγ-APC-Cy7, IL-17A-PE, Foxp3-PE, and CD19-APC (Biolegend) and RORγt-BV421 (BD Biosciences). Flow cytometry data were analyzed with FlowJo.

Mu Q., Cabana-Puig X., Mao J., Swartwout B., Abdelhamid L., Cecere T.E., Wang H., Reilly C.M, & Luo X.M. (2019). Pregnancy and lactation interfere with the response of autoimmunity to modulation of gut microbiota. Microbiome, 7, 105.

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Comprehensive Immune Cell Profiling

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Antibodies/dyes and dilutions used were: viability dye live/dead fixable-violet (L34955, Invitrogen, 1:1250), CD3-eFluor450 (48-0038, eBioscience, 1:100), CD3-PECy7 (25-0038, eBioscience, 1:100), CD4-VioGreen (130-096-900, Miltenyi Biotech, 1:50), CD8-VioGreen (130-098-062, Miltenyi Biotech, 1:50), CD8-V450 (560347, BD, 1:50), CD8-PerCP.Cy5.5/PerCP (301032, Biolegend, 1:100), CD38-APC (555462, BD, 1:50), CD69-FITC (11-0699, eBioscience, 1:40), CD161-APC (130-098-908, Miltenyi Biotech, 1:100), CD161-PE (130-099-193, Miltenyi Biotech, 1:100), IFN-γ-FITC (130-091-641, Miltenyi Biotech, 1:50), IFN-γ-APCCy7 (502529 Biolegend, 1:50), Vα7.2-PE/PeCy7/APC/FITC (351705/351711/351707/351703, Biolegend, 1:50). Granzyme B-APC (MHGB05, Invitrogen), IL-18Ra-APC (17-7183-41, eBioscience, 1:50), TNF-α-PeCy7 (502929, Biolegend, 1:100). All data was acquired on a MACSQuant (Miltenyi Biotech) or a BD FACSVerse (BD) and analyzed on FlowJo (Tree Star Inc.). Gating strategy is shown in Supplementary Fig. 12.

van Wilgenburg B., Scherwitzl I., Hutchinson E.C., Leng T., Kurioka A., Kulicke C., de Lara C., Cole S., Vasanawathana S., Limpitikul W., Malasit P., Young D., Denney L., Moore M.D., Fabris P., Giordani M.T., Oo Y.H., Laidlaw S.M., Dustin L.B., Ho L.P., Thompson F.M., Ramamurthy N., Mongkolsapaya J., Willberg C.B., Screaton G.R, & Klenerman P. (2016). MAIT cells are activated during human viral infections. Nature Communications, 7, 11653.

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Multicolor Flow Cytometry for T Cell Analysis

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The CD8-FITC, IFN-γ-APC-Cy7, CD4-FITC, CD25-APC, FoxP3-PE, lin1-FITC, CD11c-APC, HLA-DR-PE, PD-1-PE-cy7, and PD-L1-PE-cy7 mAbs were obtained from BioLegend (BioLegend, CA). Fix and Perm kit was obtained from BioLegend (BioLegend, CA). Intracellular cytokine production was detected by four-color flow cytometry [16 (link), 18 (link)], as described in previous papers. What need to be illustrated is that PMA-ionomycin stimulation causes the prominent alternation of cell membrane expression of CD4, so CD3⁺CD8⁻ cells are identified as CD4⁺ T cells [19 (link)]. After washing with PBS, the stained cells were analyzed by flow cytometry.

Ji L.S., Gao Q.T., Guo R.W., Zhang X., Zhou Z.H., Yu Z., Zhu X.J., Gao Y.T., Sun X.H., Gao Y.Q, & Li M. (2019). Immunomodulatory Effects of Combination Therapy with Bushen Formula plus Entecavir for Chronic Hepatitis B Patients. Journal of Immunology Research, 2019, 8983903.

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Cytokine Profile of Activated PBMCs

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Peripheral blood mononuclear cells were incubated overnight with 10 ng/ml LPS (Sigma-Aldrich) in the presence of GolgiPlug (1:1,000, Becton Dickinson) after pre-stimulation with IFN-γ for 2 h. The cells were then incubated with conjugated primary antibodies in phosphate-buffered saline containing 0.5% bovine serum albumine for 30 min. The antibodies used were CD3-PE, CD14-Pacfic Blue, CD16-PE-Cy7, CD20-PE, and CD56-PE (all Biolegend) at 4°C and were incubated with EDTA for 15 min followed by incubation with FACS permeabilizing solution 2 (BD Biosciences) for 15 min. Next, conjugated antibodies to TNF-α-Percp-Cy5.5, IFN-γ-APC-Cy7, IL-1β-FITC, IL-6-APC, and IL-10-APC and their respective isotype controls (all Biolegend) were added to determine intracellular cytokine production. The cells were washed and analyzed using flow cytometry (FACSCanto II, BD Biosciences) and FACSDiva software (16 (link)).

van den Bosch T.P., Caliskan K., Kraaij M.D., Constantinescu A.A., Manintveld O.C., Leenen P.J., von der Thüsen J.H., Clahsen-van Groningen M.C., Baan C.C, & Rowshani A.T. (2017). CD16+ Monocytes and Skewed Macrophage Polarization toward M2 Type Hallmark Heart Transplant Acute Cellular Rejection. Frontiers in Immunology, 8, 346.

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Profiling Immune Cells by Flow Cytometry

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Monoclonal antibodies specific for human CD3-PeCy7, CD3-APC-Cy7 (Clone HIT3a), CD4-Alexa Fluor 488 (Clone OKT4), CD8-APC-Cy7, CD8-Brilliant Violet 421 (Clone RPA-T8), IgG1-PE (Clone HP6017), IgG1 Isotype-PE (Clone MOPC−21), 41BB-PE (Clone 4B4–1), PD−1-PeCy7, PD−1-Brilliant Violet 421 (Clone EH12.2H7), CD69-APC-Cy7 (Clone FN50), IFNγ-APC-Cy7 (Clone 4S.B3), TNFα-Alexa Fluor 488 (Clone Mab11), granzyme B-APC (Clone GB−11), perforin-PerCPCy5.5 (Clone B-D48), CEA-APC (Clone ASL−32), PD-L1-Brilliant Violet 421 (Clone 29E.2A3), TruStain fcX™ (Clone 93) and viability dye Zombie Aqua (ref 423,101), were purchased from Biolegend. Samples were analyzed in a FACSCanto II cytometer (BD Bioscience) and data were analyzed using FlowJo version X.0.7 (Tree Star, Inc.).

Lopez E., Hidalgo S., Roa E., Gómez J., Hermansen Truan C., Sanders E., Carrasco C., Pacheco R., Salazar-Onfray F., Varas-Godoy M., Borgna V, & Lladser A. (2023). Preclinical evaluation of chimeric antigen receptor T cells targeting the carcinoembryonic antigen as a potential immunotherapy for gallbladder cancer. Oncoimmunology, 12(1), 2225291.

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