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Ifnγ percpcy5

Manufactured by Thermo Fisher Scientific
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IFNγ-PerCPCy5.5 is a fluorescently-labeled antibody designed for the detection and quantification of interferon-gamma (IFNγ) in flow cytometry applications. It is a conjugate of the PerCPCy5.5 fluorescent dye and an anti-IFNγ monoclonal antibody, allowing for the specific labeling and identification of IFNγ-producing cells.

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Lab products found in correlation

Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions) Eomes pe, by Thermo Fisher Scientific (2 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions) Fixation and permeabilization buffer, by BioLegend (1 mentions) Monensin solution, by BioLegend (1 mentions) Cd1a pe, by BD (1 mentions) Cd14 fitc, by Immunotools (1 mentions) Cd4 apc, by Immunotools (1 mentions) Cd40 pe, by Bio-Rad (1 mentions) Cd86 v450, by BD (1 mentions) Il 17a pe, by BioLegend (1 mentions) Foxp3 alexa fluor 647, by BD (1 mentions) Cd3 apc cy7, by BD (1 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions) Facscanto 2, by BD (1 mentions) Hla dr percp cy5, by BD (1 mentions) Cd45ra pe, by Immunotools (1 mentions) Cd45.2 v500, by BD (1 mentions) Ifn γ efluor450, by Thermo Fisher Scientific (1 mentions)

5 protocols using ifnγ percpcy5

1

Multiparameter Flow Cytometry Analysis

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Cells were stained according to the manufacturer’s recommendations with the following antibodies alone or in varying combinations, depending on the experiment: CD14-FITC, CD4-APC and CD45RA-PE (all Immunotools, Friesoythe, Germany), CD40-PE (AbD Serotec, Kidlington, UK), CD86-V450, HLA-DR-PerCPCy5.5, CD1a-PE and CD3-APC/Cy7 (all BD). For surface marker staining, PBS containing 1% FCS and 5mM EDTA (Sigma Aldrich) was used as staining buffer. For intracellular cytokine staining with IFNγ-PerCPCy5.5 (eBioscience), IL17A-PE and IL10-Brilliant Violet 421 (both BioLegend), monensin solution (BioLegend) was added to cell cultures for 6 hours before harvesting. Once harvested, cells were fixed and permeabilized using the fixation and permeabilization buffer set from BioLegend. Staining with FoxP3-Alexa Fluor 647 (BD) was performed with FoxP3 staining buffer set (eBioscience). The samples were analyzed by flow cytometry on a BD FACS Canto II.
Brencicova E., Jagger A.L., Evans H.G., Georgouli M., Laios A., Attard Montalto S., Mehra G., Spencer J., Ahmed A.A., Raju-Kankipati S., Taams L.S, & Diebold S.S. (2017). Interleukin-10 and prostaglandin E2 have complementary but distinct suppressive effects on Toll-like receptor-mediated dendritic cell activation in ovarian carcinoma. PLoS ONE, 12(4), e0175712.
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2

Multiparameter Flow Cytometry of Immune Cells

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CD45.2-V500 and TNFα-APC-Cy7 were purchased from BD Biosciences (San Jose, California). KLRG1-FITC, Eomes-PE, CD107a-PE, CD107b-PE, CD27-PE, CD127-PE-Cy5, CD127-PerCP-Cy5.5, Tbet-PerCP-Cy5.5, IFNγ-PerCP-Cy5.5, Eomes-PErCP-efluor710, CD45.1-PECy7, KLRG1-PE-Cy7, Tbet-PE-Cy7, IRF4-AlexaFluor647, CD44-AlexaFluor700, CD62L-APC-eFluor780, CD44-eFluor450, KLRG1-eFluor450, IFNγ-eFluor450, CD90.2-APC-eFluor780, CD45.1-APC-eFluor780, IL-2-PerCP-Cy5.5 were purchased from eBioscience (San Deigo, California). CD8-PE-TexasRed, GranzymeB-PE, GranzymeB-APC, Live-Dead-Violet, Live-Dead-Aqua and goat-anti-rabbit IgG-AlexaFluor647 and -AlexaFluor488 were purchased from Life Technologies (Grand Island, New York). H2Db-GP33 monomers were prepared at UMMS; LCMV-specific (H2Db-NP396, H2Db-GP276) and Influenza A PR8-OVAI-specific (H2Kb-OVA257) monomers were obtained from the NIH Tetramer Core Facility (Atlanta, Georgia). Intracellular TCF1 staining was performed using rabbit-anti-mouse TCF1 (Cell Signaling Technology, Danvers, Massachusetts) followed by staining with goat-anti-rabbit secondary (Life Technologies). Samples were analyzed on an LSRII flow cytometer (Becton Dickinson), and data were analyzed using FlowJo (Tree Star).
Nayar R., Schutten E., Bautista B., Daniels K., Prince A.L., Enos M., Brehm M.A., Swain S.L., Welsh R.M, & Berg L.J. (2014). Graded levels of IRF4 regulate CD8+ T cell differentiation and expansion, but not attrition, in response to acute virus infection. Journal of immunology (Baltimore, Md. : 1950), 192(12), 5881-5893.
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3

Multiparametric Analysis of CD4+ T Cells

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CD4+ T cells were stained with Fixable Viability Dye (eBioscience, Nieuwegein, The Netherlands), and antibodies for PlexinB2 APC and its respective isotype control (both from R&D systems, Abingdon, United Kingdom). Alternatively, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Nieuwegein, The Netherlands), and stained for IL-17A FITC, IL-22 APC, IFNγ PerCP-Cy5.5 (all from eBioscience, Nieuwegein, The Netherlands), IL-4 BV711, IL-13 PE and TNF BV421 (all from BD Biosciences, Vianen, The Netherlands). For proliferation analysis, CD4+ T cells were labeled with CellTrace Violet (1.5 µM, Thermo Fisher Scientific, Nieuwegein, The Netherlands) prior to culture. Samples were acquired on a BD LSR Fortessa (BD Biosciences), or on a BD FACSCanto (BD Biosciences, Vianen, The Netherlands) using the BD FACSDiva software (BD Biosciences, Vianen, The Netherlands). FlowJo software (BD Biosciences, Vianen, The Netherlands) was used for data analyses. All flow cytometry data is presented as the percentage of positive cells.
Carvalheiro T., Rafael-Vidal C., Malvar-Fernandez B., Lopes A.P., Pego-Reigosa J.M., Radstake T.R, & Garcia S. (2020). Semaphorin4A-Plexin D1 Axis Induces Th2 and Th17 While Represses Th1 Skewing in an Autocrine Manner. International Journal of Molecular Sciences, 21(18), 6965.
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4

Comprehensive T cell Immunophenotyping

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T cells from polarization assays were re-stimulated with 2 μL/mL cells of Cell Stimulation Cocktail (eBioscience), containing PMA/Ionomycin/Brefeldin-A/monensin, for 5 hours at 37°C. Cells were then stained with cell surface and intracellular antibodies using the Foxp3 Staining Buffer Set (eBioscience). Antibodies used for cell analyses were conjugated to FITC, PE, PerCp, or APC, and are as follows: CD4-PE, CD8-PErCp, CD25-FITC, CD44-PE, IFNγ-FITC, Annexin V-APC (from BD Biosciences),7-AAD (BD Pharmingen), granzyme B-FITC, T-bet-APC, Eomes-PE, RORγt-PE, IL-17A-APC (from eBioscience), and CD107-FITC (from BioLegend). For human CD8+ T cells, CD8-PE and IFNγ-PerCp-Cy5.5 (eBioscience) were used. Cells were analyzed on a BD FacsCalibur.
Glenn J.D., Smith M.D., Calabresi P.A, & Whartenby K.A. (2014). Mesenchymal stem cells differentially modulate effector CD8+ T cell subsets and exacerbate experimental autoimmune encephalomyelitis. Stem cells (Dayton, Ohio), 32(10), 2744-2755.
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5

Flow Cytometric Analysis of T Cell Subsets

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Lymphocytes were isolated and re-stimulated with 50 μg/mL phorbol myristate acetate + 1 μg/mL ionomycin and Brefeldin A at 37 °C and 5% CO2 for 4 h. Cells were washed and incubated with anti-Fc receptor and stained for FACS analysis using the Foxp3/transcription factor staining buffer set (eBioscience, CA, USA) with a combination of antibodies: CD45-APC (1:400 clone A20.1), CD3-A700 (1:200 clone KT3-1.1) and CD4-FITC (1:400 clone GK1.5) obtained from our monoclonal antibody facility; IFNγ-PercpCy5.5 (1:300 clone XMG1.2) and IL-17A-pacific blue (1:300 clone 17B7) purchased from eBioscience; and Live/Dead fixable dead cell stain (Life Technologies, CA, USA).
Tye H., Yu C.H., Simms L.A., de Zoete M.R., Kim M.L., Zakrzewski M., Penington J.S., Harapas C.R., Souza-Fonseca-Guimaraes F., Wockner L.F., Preaudet A., Mielke L.A., Wilcox S.A., Ogura Y., Corr S.C., Kanojia K., Kouremenos K.A., De Souza D.P., McConville M.J., Flavell R.A., Gerlic M., Kile B.T., Papenfuss A.T., Putoczki T.L., Radford-Smith G.L, & Masters S.L. (2018). NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease. Nature Communications, 9, 3728.
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