Ifn γ pacific blue

Manufactured by BioLegend

Sourced in United States

IFN-γ-Pacific Blue is a fluorescent antibody that binds to the interferon-gamma (IFN-γ) protein. It is used in flow cytometry applications to detect and quantify IFN-γ-producing cells.

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IFN-γ (422 citations)

5 protocols using ifn γ pacific blue

Comprehensive Immune Cell Profiling

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The heparinised whole blood was withdrawn from the mouse orbital sinus. Red blood cells were isolated with Easy Lyse Solution (10 mL; Leinco Technologies, USA), and then stained with Foxp3-PE, CD4-FITC, IL17A-APC, IFN-γ-Pacific Blue, IL4-PE, CD3-FITC and CD8-Pacific Blue (BioLegend, CA, USA). For graft analysis, the graft samples were homogenised and digested with collagenase (0.125%; Sigma, Missouri, USA) in a shaking water bath at 37 °C for 30 min. After centrifugation (800×g, 3 min) and resuspension, the cells were stained with the following monoclonal antibodies: CD11b-Pacific Blue, CD11c-PE/Cy7, Foxp3-PE, F4/80-PE, CD206-FITC CD4-FITC, IL17A-APC, IFN-γ-Pacific Blue, IL4-PE, CD3-FITC, and CD8-Pacific Blue (BioLegend, CA, USA). An irrelevant control monoclonal antibody was included for each fluorochrome. Finally, the cells were analysed using a BD LSR-II flow cytometer (Becton Dickinson, CA, USA). Cell Quest software (Becton Dickinson, CA, USA) was employed for data acquisition and analysis. A gate was set to exclude 99.9% of the isotype control-labelled cells.

Liu K., He Y., Yao Y., Zhang Y., Cai Z., Ru J., Zhang X., Jin X., Xu M., Li Y., Ma Q., Gao J, & Lu F. (2021). Methoxy polyethylene glycol modification promotes adipogenesis by inducing the production of regulatory T cells in xenogeneic acellular adipose matrix. Materials Today Bio, 12, 100161.

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Intracellular Cytokine and CD25+CD134+ Assays

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Intracellular cytokine assays were performed as previously described [14 (link),26 (link)]. Briefly 0.5 mL fresh sodium heparin anticoagulated blood was incubated with SEB or antigens (as above) for 2 h at 37 °C, before the addition of 10 µg/mL brefeldin A (Sigma-Aldrich), and incubated for a further 4 h, before addition of 2 mM EDTA. Aliquots were stained with CD3-PerCP-Cy5.5, CD4-PE-Cy7, CD8-APC-Cy7 and CD73-PE, lysed with FACSLyse (BD Biosciences), permeabilized with FACSPerm2 (BD Biosciences), and then incubated with anti-IL-2-APC (BD Biosciences), IFN-γ-Pacific Blue (BioLegend, San Diego, CA, USA) or –FITC (BD Biosciences) and IL-17-AF488 (BioLegend). Cells were then analyzed on an LSR II, as described above.
CD25+CD134+ (“OX40”) assays were performed as previously described [33 (link)]. Briefly, 0.25 mL IMDM was added to 0.25 mL fresh Na Hep anticoagulated blood and incubated with SEB or antigens (as above) for 40–48 h at 37 °C. Aliquots were stained with CD3-PerCP-Cy5.5, CD4-PE-Cy7, CD25-APC, CD134-FITC and CD73-PE, lysed with Optilyse C, and then analyzed on an LSR II, as described above.

Seddiki N., Zaunders J., Phetsouphanh C., Brezar V., Xu Y., McGuire H.M., Bailey M., McBride K., Hey-Cunningham W., Munier C.M., Cook L., Kent S., Lloyd A., Cameron B., Fazekas de St Groth B., Koelsch K., Danta M., Hocini H., Levy Y, & Kelleher A.D. (2021). CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection. International Journal of Molecular Sciences, 22(2), 912.

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Multiparametric Intracellular Cytokine Analysis

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For intracellular cytokine staining, CD4⁺ T cells or CD4⁺ T cell/monocyte cocultures were stimulated for 3 h in the presence of PMA (50 ng/ml; Sigma-Aldrich), ionomycin (750 ng/ml; Sigma-Aldrich), and GolgiStop (as per the manufacturer’s instructions; BD Biosciences). Cells were washed and stained with CD3-PE Cy7 (UCHT1; BioLegend) and LIVE/DEAD efluor 780 (Thermo Fisher Scientific). Cells were then fixed in 2% PFA and permeabilized with 0.5% saponin (Thermo Fisher Scientific). Cells were subsequently stained for the following cytokines: IL-10–Alexa Fluor 488 (JES3-9D7; BioLegend), IL-17A–PE (BL168; BioLegend), IFN-γ–Pacific blue (4S.B3; BioLegend), and TNF–allophycocyanin (MAb11; BioLegend).
For intranuclear staining of IKZF3, cells were fixed and permeabilized with FOXP3 staining buffer (BioLegend) for 15 min at room temperature before being stained for CD3-PE Cy7, IL-10–Alexa Fluor 488, IL-17A–PE, IFN-γ–Pacific blue, TNF-BV605 (MAb11; BioLegend), and either IKZF3-Alexa Fluor 647 (EPR9342[B]; Abcam) or isotype control (EPR25A; Abcam) for 30 min. Standard gating strategy for intracellular cytokine staining is shown in Supplemental Fig. 1A–C.

Ridley M.L., Fleskens V., Roberts C.A., Lalnunhlimi S., Alnesf A., O’Byrne A.M., Steel K.J., Povoleri G.A., Sumner J., Lavender P, & Taams L.S. (2020). IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4+ T Cells. The Journal of Immunology Author Choice, 204(11), 2940-2948.

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NK Cell-Mediated Tumor Cytotoxicity Assay

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NK cells were harvested, washed with their culture media and co-cultured with tumor targets at a 1:1 effector-to-target ratio. Different combinations of NK cells, tumors, stimulation molecules and antibodies were incubated with anti-CD107a PE (BD Biosciences, Franklin Lakes, NJ, 555801) for 1 hour. Following incubation, 1:1000 Golgi Stop and 1:1000 Golgi Plug solutions (BD Biosciences, 554724, 555029) were added and the cells incubated for an additional 2 hours. Cells were then counterstained with Live/Dead Fixable Aqua Dye (Thermo Fisher Scientific, L34957), IgG1 Isotype APC (BD Biosciences, 551442) and CD56-APC (BD Biosciences, 555518) for 30 min at 4°C. After staining, the cells were washed with buffer containing DPBS (Lonza, Basel, Switzerland, 17–512), 2% standard FBS (Life Technologies, 10100147), 0.1% NaN3 (Sigma Aldrich, 71289) and then fixed with BD Cytofix (BD Biosciences, 554655) for 20 min at 4C. Next, cells were washed and permeabilized using BD Perm/Wash (BD Biosciences, 554723) for 15 min at 4C. Afterwards, cells were stained for 30 min with IFN-γ Pacific Blue (Biolegend, San Diego, CA, 502521). After washing, cells were analyzed.

Zhu H., Blum R.H., Bernareggi D., Ask E.H., Wu Z., Hoel H.J., Meng Z., Wu C., Guan K.L., Malmberg K.J, & Kaufman D.S. (2020). Metabolic reprograming via deletion of CISH in human iPSC-derived NK cells promotes in vivo persistence and enhances anti-tumor activity. Cell stem cell, 27(2), 224-237.e6.

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Phenotyping of Myeloid-Derived Suppressor Cells in PBMC

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PBMC were isolated from fresh peripheral blood by using density centrifugation (Ficoll-Paque; GE Healthcare). For the analysis of MDSC, PBMC were surface labeled with anti-CD14-BD Horizon V450 (BD Biosciences), anti-HLA-DR-Alexa Flour 700 (Biolegend), anti-CD11b-FITC (eBioscience), anti-CD33-PE-Cy7 (Biolegend), anti-CD15-APC (eBioscience). The gating strategy is depicted in S1 Fig. Expression of CD3ζ was analyzed on PBMC by intracellular staining using a fixation/permeabilization buffer kit (eBioscience) with appropriate mAb anti-CD3ζ-PE (Exbio). For the analysis of IFN-γ and IL-17A producing cells, PBMC were stimulated with PMA (50 ng/ml; Sigma-Aldrich) plus ionomycin (1 mg/ml; Sigma-Aldrich) for 4h in the presence of brefeldin A (5 mg/ml; Biolegend). Intracellular IFN-γ and IL-17A staining were assessed using a fixation/permeabilization buffer kit (eBioscience) with appropriate mAb IFN-γ-Pacific Blue (Biolegend) and anti-IL-17-A647 (Biolegend) and measured by flow cytometry. Data were acquired by LSRFortessa and LSR II flow cytometers (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Grohová A., Dáňová K., Adkins I., Šumník Z., Petruželková L., Obermannová B., Koloušková S., Špíšek R, & Palová-Jelínková L. (2020). Myeloid - derived suppressor cells in Type 1 diabetes are an expanded population exhibiting diverse T-cell suppressor mechanisms. PLoS ONE, 15(11), e0242092.

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