Ifn γ apc xmg1

Manufactured by BioLegend

IFN-γ-APC (XMG1.2) is a fluorochrome-conjugated antibody that binds to interferon-gamma (IFN-γ), a cytokine involved in immune response. This product is suitable for flow cytometry applications.

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IFN-γ (APC; clone XMG1.2; Anti-IFN-γ–APC (clone XMG1.2, APC-labeled anti-IFN-γ (XMG1.2) APC anti-mouse IFN-γ clone XMG1.2 IFN-γ-APC IFN-γ

5 protocols using ifn γ apc xmg1

Multiparametric T Cell Analysis

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Analyses were performed using the following antibodies: CD3-FITC (145-2C11, Biolegend), CD4-BV750 (GK1.5, Biolegend), CD8-BV786 (53-6.7, Biolegend), CD62L-BV510 (MEL-14, Biolegend), CD44-PE/Cy5 (IM7, Biolegend), CD107a-BV711 (1D4B, Biolegend), CD154-PE (MR1, BD), Gmzb-AF700 (QA16A02, Biolegend), IFN-γ-APC (XMG1.2, Biolegend) and TNF-α-BB700 (MP6-XT22, BD). In brief, splenocytes isolated from experimental mice were cultured in the presence of phorbol 12-myristate 13-acetate (PMA) (1.9 nM) and ionomycin (0.08 mg/ml) solution at 37°C for 4 h. After washing, 2 × 10⁶ cells were stained with FVS780 (viability dye) followed by treatment with antibodies for surface antigens (Anti-CD3, CD4, CD8, CD62L, CD44, CD107a, and CD154) at 4°C for 30 min. After washing, cells were fixed and permeabilized with BD Cytofix/Cytoperm, and then stained with the antibodies against the intracellular antigens (Gzmb, IFN-γ, and TNF-α) for 20 min at 22°C. Stained cells were separated using a BD FACSymphony™ Cell Analyzer and one million events were collected for each sample. The analysis was performed in FlowJo software version 10.6.2.

Amano T., Yu H., Amano M., Leyder E., Badiola M., Ray P., Kim J., Ko A.C., Achour A., Weng N.P., Kochba E., Levin Y, & Ko M.S. (2023). Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity. iScience, 26(4), 106335.

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Multiparameter Flow Cytometry Immunophenotyping

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The following antibodies were used: CD11b-APC-Cy7 (M1/70), CD19-Biotin (6D5), TCRβ-Biotin (H57-597), NK1.1-Biotin (PK136), Ly6C-BV570 (HK1.4), Ly6G-PerCp-Cy5.5 (1A8), and TCRβ-Pacific Blue (H57-597) from Biolegend (San Diego, CA) CD8α-PE-Cy7 (53-6.7), CD4-Alexa 700 (RM4-5), and CD19-FITC (1D3) from BD Pharmingen (San Diego, CA); CD49b (DX5) from eBioscience (San Diego, CA) and MHC-II-Biotin (produced in house from hybridoma M5/114). The secondary Streptavidin-PE-CF594 from BD Horizon was used with biotin-conjugated primary antibodies. Antibody for detection of intracellular IFN-γ was IFN-γ-APC (XMG1.2) and the isotype control was Rat IgG1, κ-APC (RTK2071) (both from Biolegend). PE-labeled CD1d-tetramers loaded with α-galactosyl ceramide (αGalCer) (PBS57) were provided by the NIH Tetramer Facility and used to detect NKT cells.

Fernández-Santoscoy M., Wenzel U.A., Yrlid U., Cardell S., Bäckhed F, & Wick M.J. (2015). The Gut Microbiota Reduces Colonization of the Mesenteric Lymph Nodes and IL-12-Independent IFN-γ Production During Salmonella Infection. Frontiers in Cellular and Infection Microbiology, 5, 93.

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Multiparametric Flow Cytometry of Immune Cells

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Cells were washed with FACS buffer (HBSS (Corning, without Calcium, Magnesium and Phenol Red) containing 0.2% BSA (Sigma A3059)and 0.05% Azide(Sigma S2002)), treated with anti-FcR (24G2, Leino C381), and then stained with specific antibodies at 4 °C for 30 min. We use anti–mouse CD4-FITC (RM4-4, Biolegend 100510), CD4-BV785(RM4-4, Biolegend 100552), CD8α-PE-Cy7 (53-6.7, Biolegend 100722), B220-AF594 (RA3-6B2, Biolegend 103254), CD11c-PE (N418, Biolegend 117308), MHCII-BV421 (M5/114.15.2, Biolegend 107632), CD40-APC (3/23, Biolegend 124612), IFN-γ-APC (XMG1.2, Biolegend 505810), anti-IL17A-BV785 (TC11-18H10.1, Biolegend 506928), GM-CSF-PE (MP1-22E9, Biolegend 505406) antibodies were purchased from Biolegend. For intracellular cytokine staining, mononuclear cells from LNs and CNS were stimulated overnight with rhMOG (10μg/ml), with monensin (Biolegend 420701) added in the last four hours, and cells were fixed and permeabilized with the BD Fix/Perm kit (BD 554715) according to the manufacturer’s instructions and then stained with intracellular antibodies for 30 min. Dead cells were excluded using Zombie Aqua™ Fixable Viability Kit (Biolegend 423102). Data were collected with a FACS LSR II or FACS Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software.

Lu Y., Xu M., Dorrier C.E., Zhang R., Mayer C.T., Wagner D., McGavern D.B, & Hodes R.J. (2022). CD40 drives CNS autoimmune disease by inducing complementary effector programs via B cells and dendritic cells. Journal of immunology (Baltimore, Md. : 1950), 209(11), 2083-2092.

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Multicolor Flow Cytometry Panel for Murine Cells

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Murine cells were blocked with rat anti-mouse CD16/32 (0.5 μg per well, eBioscience) and stained with respective antibodies at diluations according to manufacturers’ protocols: CD45-BV510 (30-F11, BioLegend), CD4–antigen-presenting cell (APC), CD4-BV510 (both RM4–5, BioLegend), CD8-Percp-Cy5.5 (53–7.7, eBioscience), CD3-FITC, CD3-BV421 (both 17A2, BioLegend), IFNγ-PE, IFNγ-APC (XMG1.2, BioLegend), HLA-A2-APC (BB7.2, BioLegend), HLA-DR-Percp eFl710 (L243, BioLegend), Ly6C-APC (KK1.4, BioLegend), CD11B-PE-Dazzle 594 (M1/70, BioLegend), Cxcl9-PE (MIG-2F5.5, BioLegend), I-A/I-E-AF700 (M5/114.15.2, BioLegend), CD11C-BV785 (N418, BioLegend). eFluor 780 fixable viability dye (eBioscience) was used according to manufacturer's protocol to exclude dead cells. For intracellular staining, cells were incubated at 37°C with 5 μg/mL Brefeldin A (Sigma) and respective stimulus (10 μg/mL for peptides, see also Elispot section) for 4 to 6 hours. Intracellular staining was performed using eBioscience Intracellular Fixation & Permeabilization Buffer Set for cytokines according to manufacturer's protocol. Nonfixed samples were acquired immediately, and fixed samples were acquired within 48 hours on a FACS Canto II (BD Biosciences) or a BD AriaII.

Kilian M., Friedrich M., Sanghvi K., Green E., Pusch S., Kawauchi D., Löwer M., Sonner J.K., Krämer C., Zaman J., Jung S., Breckwoldt M.O., Willimsky G., Eichmüller S.B., von Deimling A., Wick W., Sahm F., Platten M, & Bunse L. (2021). T-cell Receptor Therapy Targeting Mutant Capicua Transcriptional Repressor in Experimental Gliomas. Clinical Cancer Research, 28(2), 378-389.

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Multiparametric Phenotyping of CD8+ T Cells

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Gp_33‐41 peptide (KAVYNFATM) and gp_61‐80 (GLNGPDIYKGVYQFKSVEFD) peptides were purchased from Mimotopes. PE‐conjugated gp33‐MHC class I tetramer (H‐2Db/gp33‐41) was kindly provided by the NIH tetramer core facility. For exclusion of dead cells, efluor780 (eBioscience) or ZombieAqua (BioLegend) was used. Antibodies used were as follows: PD‐1‐FITC (J43; Thermo Fisher Scientific), HVEM‐APC (LH1; eBioscience), CD19‐APC‐Cy7 (6D5; BioLegend), CD4‐PE (RM4‐5; BD Bioscience), CD4‐PerCP‐Cy5.5 (RM4‐5; BioLegend), CD8a‐FITC (53‐6.7, eBioscience), CD8a‐PE‐Cy7 (53‐6.7; BioLegend), CD8a‐APC‐Cy7 (53‐6.7; BioLegend), CD8a‐PE (53‐6.7; BD Bioscience), IFNγ‐APC (XMG1.2; BioLegend), CD127‐APC (SB/199; BioLegend), CD25‐PE (PC61; eBioscience), Ki‐67‐PE (16A8; BioLegend), KLRG‐1‐PerCP‐eFluor710 (2F1; eBioscience), CD62L‐BV421 (MEL‐14; BioLegend), CD45.1‐PerCP‐Cy5.5 (A20; BioLegend), TNFα‐PE (MP6‐XT22; BD Bioscience), and TNFα‐FITC (MP6‐XT22; BioLegend).

Diethelm P., Schmitz I., Iten I., Kisielow J., Matsushita M, & Kopf M. (2022). LCMV induced downregulation of HVEM on antiviral T cells is critical for an efficient effector response. European Journal of Immunology, 52(6), 924-935.

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