Ifn γ pe cy7

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IFN-γ-PE-Cy7 is a conjugate of the cytokine interferon-gamma (IFN-γ) and the fluorescent dye PE-Cy7. It is used for the detection and quantification of IFN-γ-producing cells in flow cytometry experiments.

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Close spelling variants of this product

Anti-mouse IFN-γ-PE-Cy7 (2 citations) Anti-IFN-γ-PE-Cy7 (17 citations) PE-Cy7 anti-human IFN-γ (3 citations) IFN-γ–PE-Cy7 (4S.B3; (2 citations) PE/Cy7-conjugated anti-IFN-γ (clone B27, (2 citations) PE-Cy7-anti-IFN-γ (clone 4S.B3; (2 citations) PE-Cy7-conjugated anti-IFN-γ (4 citations) IFN-γ-PE (37 citations) IFN-γ (422 citations)

20 protocols using ifn γ pe cy7

Comprehensive Immunophenotyping Panel

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Alexa Fluor 488 NK1.1 (PK136), Biotin-CD4 (GK1.5), Alexa Fluor 488-CD4 (GK1.5), APC-eFluor 780-CD4 (GK1.5), FITC-CD3ε (17A2), Alexa Fluor 647-CD3ε (17A2), Alexa Fluor 488-CD206, Brilliant Violet 421-CD3ε (17A2), FITC-F4/80, Alexa Fluor 488-CD3ε (145-2C11), Alexa Fluor 647 CD49b (DX5), Pacific Blue-CD49b (DX5), PE-NK1.1 (PK136), Brilliant Violet 421 NK1.1 (PK136), PE/Cy5-CD4 (GK1.5), APC-TCRγδ (GL3), Biotin-IFN-γRα (2E2), Brilliant Violet 421-CD25 (PC61), PE-CD25 (PC61), Biotin-CD122 (5H4), PE/Cy7-IFN-γ (XMG1.2), PE-GM-CSF (MP1-22E9), Pacific Blue-TNF-α (MP6-XT22), PercCP/Cy5.5-CD69, Biotin-BrdU (Bu20a), FITC-Ki67 (16A8), Alexa Fluor 647-streptavidin, APC-Cy7-Streptavidin, PE-Cy7-Streptavidin, Brilliant violet 421-CD62L (MEL-14) and rabbit polyclonal anti-Asialo-GM1 (aGM) antibodies were purchased from Biolegend (San Diego, CA). APC-RORγt (AFKJS-9), APC-T-bet (4B10) and PE/Cy7-T-bet (4B10) were procured from eBioscience (San Diego, CA). Anti-NK1.1 (PK136), anti-F4/80 (Cl:A3–1), anti-Gr1 (RB6-8C5) and isotype control antibodies for in vivo use were procured from BioXcell (West Lebanon, NH). Alexa Fluor 647-labeled CD1d tetramer (mCD1d, PBS-57) and unloaded control tetramer were received from NIH Tetramer Core Facility (Atlanta, GA). 5-Bromo-2′-deoxyuridine (BrdU) was purchased from Sigma-Aldrich.

Paul S., Chhatar S., Mishra A, & Lal G. (2019). Natural killer T cell activation increases iNOS+CD206- M1 macrophage and controls the growth of solid tumor. Journal for Immunotherapy of Cancer, 7, 208.

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Comprehensive Cell Line Validation and Inhibitor Characterization

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All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), independently validated by short tandem repeat (STR) DNA fingerprinting, and tested negative for mycoplasma infection. NCI-H157, A549, NCI-H1299, HeLa, BT549, MDA-MB-231, CT26, B16-F10, MC-38, LLC and 293T cells were cultured in RPMI 1640 or DMEM media, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a humidified atmosphere with 5% CO₂. Inhibitors for ATM (KU-60019, AZD1390, AZD0156), STING (H151), p-TBK1 (GSK8612), p-STAT1 (fludarabine) were purchased from Selleck (Houston, TX, USA). Human IFNβ and Galectin-9 ELISA assay Kits were purchased from R&D SYSTEMS (Minneapolis, MN, USA). Anti-human Gal-9 antibody was purchased from BioRad (Hercules, CA, USA). Anti-mouse Gal-9 antibody and IgG isotype control were purchased from BioXCell (Lebanon, NH, USA). Antibodies against cGAS, STING, p-STING(Ser366), p-TKB1(Ser172), TKB1, p-STAT1(Tyr701), STAT1 and Actin were purchased from Cell Signaling Technology (Cambridge, MA, USA). Anti-mouse fluorochrome-conjugated antibodies including FITC-CD4, Percp/Cy5.5-CD8, AF700-CD3, APC/Cy7-CD45, PE/Cy7-IFNγ antibodies were purchased from BioLegend (San Diego, CA, USA).

Zheng S., Song J., Linghu D., Yang R., Liu B., Xue Z., Chen Q., Liu C., Zhong D., Hung M.C, & Sun L. (2023). Galectin-9 blockade synergizes with ATM inhibition to induce potent anti-tumor immunity. International Journal of Biological Sciences, 19(3), 981-993.

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Intracellular Cytokine Staining of LP Cells

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LP cells were isolated from mice at day 0, day 12 of AOM/DSS or at the tumor endpoint (typically day 60). For intracellular staining, cells were incubated for 4 hours at 37°C with 1) GolgiStop (monensin, BD), 100 ng/ml PMA (Sigma) and 1000 ng/ml ionomycin, or 2) with GolgiStop alone (“no stim”). Cells were surface stained, fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), and then incubated with fluorochrome-conjugated antibodies against PE-Cy7 IFNγ (Biolegend), FITC IL17A (Biolegend), and PE Foxp3 (eBioscience). Samples were analyzed by a BD LSRFortessa, FACSCanto II, or FACSAria II flow cytometer. Foxp3 staining was done on unstimulated cells.

Yu A.I., Zhao L., Eaton K.A., Ho S., Chen J., Poe S., Becker J., Gonzalez A., McKinstry D., Hasso M., Mendoza-Castrejon J., Whitfield J., Koumpouras C., Schloss P.D., Martens E.C, & Chen G.Y. (2020). Gut Microbiota Modulate CD8 T Cell Responses to Influence Colitis-Associated Tumorigenesis. Cell reports, 31(1), 107471.

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Multiparametric Immunophenotyping of MAIT Cells

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Single-cell suspensions were stained with the fluorescent-labeled antibodies (anti-human series, Biolegend), including FITC-CD3(OKT3), PE-TCR/γδ(B1), APC- TCRVα7.2(3C10), Brilliant Violet 510-CD161(HP-3G10), PE/Cy7-IFN-γ(B27), PE/Cy7-IL-2(MQ1-17H12), Brilliant Violet 421-TNF-α(MAb11), Brilliant Violet 421-IL-17A(BL168), PE/Cy7-CD4(RPA-T4), APC/Cyanine7-CD8(SK1). Isotype controls were used to determine cut-off levels for positive staining. Data were acquired using Flow Cytometer (Backman, America) and analyzed by FlowJo_V10 software.

Shao C., Zhu C., Zhu Y., Hao J., Li Y., Hu H., Si L., Zhong F., Wang X, & Wang H. (2021). Decrease of peripheral blood mucosal‐associated invariant T cells and impaired serum Granzyme-B production in patients with gastric cancer. Cell & Bioscience, 11, 12.

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Cytokine Profiling of Activated CD4 and CD8 T Cells

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Thawed CBMCs (1×10⁶ cells/condition) were stimulated with phorbol 12-myristate 13-acetate (0.1 μg/mL) and ionomycin (1.0 μg/mL) or media alone (R10; 10% fetal bovine serum) for 5 h at 37 °C. Brefeldin A and monensin (BD Pharmingen) were added after 1 h of incubation at a final concentration of 10 mg/mL. After 5 h, cells were washed, fixed and permeabilized, as per the manufacturer’s instructions (FoxP3 Fix/Perm kit; eBiosciences), then stained with PE-Cy5.5 CD3 (Clone SK7), PE-e610 TNFα (clone MAB11, eBioscience), BV570 CD4 (Clone RPA-T4), BV711 CD8 (clone RPA-T8), BV650 CD45RA (clone HI100), BV605 CD45RO (clone UCHL1), BV510 γδTCR (clone B1), BV510 CD14 (Clone M5E2), BV510 CD19 (Clone HIB19), PE-Cy7 IFN-γ (Clone 4 S.B.3), BV421 IL-2 (clone MQ1-17H12), AF488 IL-8 (clone MQ1-17H12, Biolegend), and LIVE/DEAD aqua amine (Invitrogen). Data were collected on an LSR II (BD) and analyzed with FlowJo software (Treestar). Cord blood CD4 and CD8 T cell cytokine responses to mitogen stimulation were gated on non-naive effector populations by expression of CCR7 and CD45RO (as in Supplementary Fig. 1) for all except IL-8, which was gated on total CD4 T cells.

Prahl M., Odorizzi P., Gingrich D., Muhindo M., McIntyre T., Budker R., Jagannathan P., Farrington L., Nalubega M., Nankya F., Sikyomu E., Musinguzi K., Naluwu K., Auma A., Kakuru A., Kamya M.R., Dorsey G., Aweeka F, & Feeney M.E. (2021). Exposure to pesticides in utero impacts the fetal immune system and response to vaccination in infancy. Nature Communications, 12, 132.

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Multiparametric Flow Cytometry Analysis

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Single-cell suspensions were prepared from the spleen and VAT and stained with PE-MHC-II (107613, Biolegend), APC-cy7-CD11c (117323, Biolegend), FITC-CD64 (139315, Biolegend), Percp-cy5.5-CD45.2 (109827, Biolegend), APC-CD45 (147707, Biolegend), FITC-CD4 (100405, Biolegend), APC-CD8 (126613, Biolegend), PE-cy7-CD62L (104417, Biolegend), Percp-cy5.5-CD44 (103031, Biolegend), APC-cy7-FVD, or Pacific blue-DAPI. Intracellular staining was performed using PE-Foxp3 (12-5773-82, eBioscience) and antibodies against cytokines, including BV421-CD8, PE-cy7-IFN-γ (505825, Biolegend), Percp-cy5.5-IL-17A (506919, Biolegend), and APC-IL-4 (504105, Biolegend). Cells from the spleen and VAT were treated with phorbol myristate acetate (PMA, 50 ng/mL) and ionomycin (500 ng/mL) (Sigma-Aldrich) for 4.5 h. Then the cytokines in specific cell populations were analyzed. The cells were stained at 4 °C for 30 min or 1 h for surface and intracellular staining, respectively, followed by detection using the FACS Canto II (BD Biosciences) system and analysis of the data using FlowJo (Tree Star, version 10.0).

Sun Y., Wang B., Hu Q., Zhang H., Lai X., Wang T., Zhao C., Wang J., Zhang X., Niu Q., He B., Jiang E., Shi M., Feng X, & Luo Y. (2023). Loss of Lkb1 in CD11c+ myeloid cells protects mice from diet-induced obesity while enhancing glucose intolerance and IL-17/IFN-γ imbalance. Cellular and Molecular Life Sciences, 80(3), 63.

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Multicolor Flow Cytometry for T and B Cell Subsets

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PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4⁺/CD8⁺ T cell ratio (Supplementary Table 1). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.

Hartzell S., Bin S., Cantarelli C., Haverly M., Manrique J., Angeletti A., Manna G.L., Murphy B., Zhang W., Levitsky J., Gallon L., Yu S.M, & Cravedi P. (2020). Kidney Failure Associates With T Cell Exhaustion and Imbalanced Follicular Helper T Cells. Frontiers in Immunology, 11, 583702.

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Comprehensive Immune Cell Profiling

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The cells were extracted from lymph nodes, spleen, or colon tissue digested by collagenase. Isolated cells were stained with following antibodies: CD8-PE-CY7 (Thermo Scientific), CD4-APC-Fire™ 750 (BioLegend), IFN-γ-FITC (BioLegend), IL-17-APC (BioLegend), FOXP3-PE (BioLegend), CD19-PE (BioLegend), NK1.1-FITC (BioLegend), CD11B-APC (BioLegend), Gr-1-FITC (BioLegend), F4/80-PE (BioLegend), CD11C-APC-CY7 (BioLegend), MHC-II-PE/Dazzle™ 594 (BioLegend), CD8-APC (BioLegend), Ki67-PerCP-CY5.5 (BioLegend), Annexin V-FITC (BioLegend), CD103-PE (BioLegend), CD69-PE-CY7 (BioLegend), CD44-PE-CY7 (BioLegend), CD62L-FITC (BioLegend), CD107-FITC (BioLegend), IFN-γ-APC (BioLegend), CD107-PE (BioLegend), IFN-γ-PE (BioLegend), CD3-FITC (BioLegend), CD3-APC/Fire™ 750 (BioLegend), CD8-APC (BioLegend), IFN-γ-PE-CY7 (BioLegend), CD69-APC/Fire™ 750 (BioLegend), Tetramer-SIINFEKL-PE (MBL), p-JNK-PE (Cell Signaling Technology), p-IRAK4-Alexa Fluor 488 (Cell Signaling Technology). Intracellular staining was carried out using the Cytofix/Cytoperm kit (BD Pharmingen) following a 4 h restimulation by PMA/ionomycin (Sigma) in the presence of GolgiPlug (BD Pharmingen). Flow cytometric data acquisition was performed on a NovoExpress flow cytometer and analyzed with NovoExpress software (ACEA Biosciences).

Wang Z., Zeng F.L., Hu Y.W., Wang X.Y., Zhao F.L., Zhou P., Hu J., Xiao Y.Y., Hu Z.L., Guo M.F., Wei X.Q., Liu X., Huang N.Y., Zhang J., Chen S.W., Cheng J., Zheng H.P., Zhou H., Zhao Q.X., Zhang C., Hao Y., Zou S., Gui Y.Y., Yu J.D., Gu L.N., Yue C.C., Zhang H.Z., Wu W.L., Zhou Y.F., Zhou X.K., Shen G.B., Teng X, & Li J. (2022). Interleukin-37 promotes colitis-associated carcinogenesis via SIGIRR-mediated cytotoxic T cells dysfunction. Signal Transduction and Targeted Therapy, 7, 19.

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Multiparameter Flow Cytometry of Immune Cells

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Cells in BALF were stained with CD45-FITC (clone 30-F11), CD11b-APC (clone M1/70), CD11c-PE (clone N418), F4/80-PE-CY7 (clone BM8), and Ly6G-BV421 (clone 1A8, BioLegend) in the presence of an Fc blocker (CD16/CD32, BD Biosciences). A total of 2 × 10⁶ single lung cells were stimulated with cell stimulation cocktail and protein transport inhibitor cocktail (Cat#00-4970-03 and #00-4980-93, eBioscience) in IMDM medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin for 4 h in a 37°C incubator with 5% CO₂. The cells were incubated with a Zombie NIR™ Fixable Viability Kit (Cat#423105, BioLegend) at room temperature for 15 min and then stained with antibodies to extracellular antigens in the presence of Fc blocker at 4°C for 25 min. Intracellular and nuclear staining was performed with the Foxp3/Transcription buffer set (Cat# 00-5523-00, eBiosciences). Data were acquired using LSRFortessa and FACSDiva software (BD Biosciences) or Attune NxT 3 L-BRV and Attune NxT software (Thermo Fisher Scientific) and analyzed using FlowJo 10.7.2 (TreeStar, Ashland). The following antibodies were used: CD4-FITC (clone GK1.5), TCR-BV421 (clone H57-597), Foxp3-AF647 (clone MF23), IL-10-PE (JES5-16E3, BD Biosciences), IFN-γ-PE-Cy7 (clone XMG1.2), IL-4-BV605 (clone 11B11), IL-17a-BV510 (clone TC11-18H10.1, Biolegend), and IL-13-PE-eFluor 610 (eBiosciences).

Li J.D, & Yin J. (2023). Interleukin-10-alveolar macrophage cell membrane-coated nanoparticles alleviate airway inflammation and regulate Th17/regulatory T cell balance in a mouse model. Frontiers in Immunology, 14, 1186393.

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Multiparametric Flow Cytometry of Murine and Human Samples

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The antibodies used for murine experiments were CD11b-FITC, Ly6G-PECy7, Ly6C-APC, CD3-PECy7, CD4-APC, CD8-FITC, IFNγ-PECy7 and isotype controls. Tumor load in the 5T33MM model was determined by the use of anti-idiotype (3H2) monoclonal antibody (IgG1) and APC-labeled rat anti-mouse IgG1 antibody (secondary step). The development of 3H2 was described previously [23 (link)]. The following antibodies were used for human experiments: CD4/CD8-Alexa Fluor^®647, CD15-Pacific Blue™, CD33-PE, CD11b-PECy7, HLA-DR-FITC, CD14-APC and isotype controls. All antibodies (except 3H2 and IFNγ-PECy7) were obtained from Biolegend (Biolegend, San Diego, CA, USA). IFNγ-PECy7 was derived from Becton Dickinson. Flow cytometric data were acquired using FACS Canto or Fortessa (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed by FACS Diva Software (BD).

De Veirman K., Van Ginderachter J.A., Lub S., De Beule N., Thielemans K., Bautmans I., Oyajobi B.O., De Bruyne E., Menu E., Lemaire M., Van Riet I., Vanderkerken K, & Van Valckenborgh E. (2015). Multiple myeloma induces Mcl-1 expression and survival of myeloid-derived suppressor cells. Oncotarget, 6(12), 10532-10547.

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