Ifn γ xmg1.2

Manufactured by BioLegend

Sourced in United States

IFN-γ (XMG1.2) is a monoclonal antibody that binds to and neutralizes interferon-gamma (IFN-γ). It is commonly used in research applications to study the role of IFN-γ in various biological processes.

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Lab products found in correlation

Lsrfortessa, by BD (5 mentions) Cd69 h1.2f3, by BioLegend (4 mentions) Cd4 rm4 5, by BioLegend (4 mentions) Il 17a tc11 18h10.1, by BioLegend (4 mentions) Facscanto 2, by BD (4 mentions) Golgiplug, by BD (4 mentions) Gr 1 rb6 8c5, by BioLegend (3 mentions) Cd45 30 f11, by BioLegend (3 mentions) Ionomycin, by Merck Group (3 mentions) Tnf α mp6 xt22, by BioLegend (3 mentions) Tnf mp6 xt22, by BioLegend (3 mentions) Cd4 gk1, by BioLegend (3 mentions) Cd11b m1 70, by Thermo Fisher Scientific (3 mentions) Cytofix cytoperm, by BD (3 mentions) Tnfα mp6 xt22, by Thermo Fisher Scientific (3 mentions) Cytofix cytoperm kit, by BD (3 mentions) Rm4 4, by BioLegend (2 mentions) Il 4 11b11, by BioLegend (2 mentions) F4 80 bm8, by BioLegend (2 mentions) Lsrii flow cytometer, by BD (2 mentions)

Spelling variants of this product

IFN-γ (422 citations) Anti-mouse IFN-γ (XMG1.2) (6 citations) IFN-γ-PE (clone XMG1.2; (4 citations) Anti-mouse IFN-γ Ab (XMG1.2) (4 citations) Anti-IFN-γ-FITC (XMG1.2) (2 citations) IFN-γ-PE (XMG1.2; (2 citations) IFN-γ (APC; clone XMG1.2; (2 citations) APC-labeled anti-IFN-γ (XMG1.2) (2 citations)

44 protocols using ifn γ xmg1.2

Murine NKT Cell Activation Protocol

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C57BL/6, NOD/LtDvs and BALB/c mice were purchased from the Jackson Laboratory. All in vivo experiments were approved by the IACUCs at the Jackson Laboratory, the University of Vermont and TGA Sciences (NKT Therapeutics Laboratory). Animals were euthanized by CO2 asphyxiation.
NKT14 and NKT14m were expressed in CHO cell lines and purified at Eureka Therapeutics. PBS57-loaded CD1d Tetramer was kindly provided by the NIH Tetramer facility. Fluorochrome labeled monoclonal antibodies targeting CD3 (17A2), CD4 (RM-4.4), CD8 (53–6.7), CD25 (PC61), CD69 (H1.2F3), IL-4 (11B11), IFN-γ (XMG1.2) and murine IgG2a FC (RMG2a.62) were purchased from Biolegend.

Scheuplein F., Lamont D.J., Poynter M.E., Boyson J.E., Serreze D., Lundblad L.K., Mashal R, & Schaub R. (2015). Mouse Invariant Monoclonal Antibody NKT14: A Novel Tool to Manipulate iNKT Cell Function In Vivo. PLoS ONE, 10(10), e0140729.

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Tumor-Infiltrating Immune Cell Analysis

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Twenty-four hours after the last exercise session, mice were anesthetized, and tumors were harvested, and samples were processed as previously described (3 (link)). Flow cytometry data were obtained using an LSRII flow cytometer (BD Biosciences) and/or Aurora (Cytek) and analyzed with FlowJo software. The double/aggregated cells were gated out using forward scatter area (FSC-A) versus forward scatter width (FSC-W) and side scatter area (SSC-A) versus side scatter width (SSC-W). Different fluorophores conjugated with the following mAb were used: CD45 (30-F11, Biolegend), TCRβ chain (H57-597, Biolegend), CD4 (RM4-5, Biolegend), FOXP3 (150D, Biolegend), CD8a (53-6.7, Biolegend), CD11c (N418, Biolegend), Gr1 (RB6-8C5, Biolegend), MHC-II (M5/114.15.2, BD Biosciences) CD11b (M1/70, Biolegend), B220 (RA3-6B2, Biolegend), F4/80 (BM8, Biolegend), IFNγ (XMG1.2, Biolegend), Granzyme B (GB11, Biolegend), Ki67 (16A8, Biolegend), CD62L (W18021D, Biolegend), CD44 (NIM-R8, Biolegend), CD69 (H1.2F3, Biolegend), CXCR3 (173, Biolegend), IL15Rα (6B4C88, Biolegend), IL6Rα (D7715A7, Biolegend), CD247 (Biolegend, 10F.9G2).

Gomes-Santos I.L., Kumar A.S., Hausmann F., Meyer M.N., Shiferaw S.Z., Amoozgar Z., Jain R.K, & Fukumura D. (2024). Exercise intensity governs tumor control in mice with breast cancer. Frontiers in Immunology, 15, 1339232.

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Intracellular Cytokine Staining and Analysis

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For intracellular cytokine staining, cells were restimulated for 4 h with 50 ng/ml phorbol 12-myristate 13-acetate and 1 µM ionomycin (Sigma-Aldrich) in the presence of GolgiStop (BD). Cells were fixed with Cytofix/Cytoperm Fixation and Permeabilization Solution (BD) and stained for intracellular cytokines. Cells were analyzed on a FACSCalibur (BD). Antibodies to the following antigens were used: CD201 (EPCR; eBio1560; eBioscience), CD4 (RM4-5; BioLegend), IL-17A (TC11-18H10.1; BioLegend), IFN-γ (XMG1.2; BioLegend), CD62L (MEL-14; BioLegend), CD44 (IM7; BioLegend), IL-1R (JAMA-147; BioLegend), and GM-CSF (MP1-22E9; BioLegend). All flow cytometric data were analyzed using FlowJo Software (Tree Star). IL-17A concentration in the supernatant was determined by ELISA using standard protocols.

Kishi Y., Kondo T., Xiao S., Yosef N., Gaublomme J., Wu C., Wang C., Chihara N., Regev A., Joller N, & Kuchroo V.K. (2016). Protein C receptor (PROCR) is a negative regulator of Th17 pathogenicity. The Journal of Experimental Medicine, 213(11), 2489-2501.

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Multiparametric Flow Cytometry Panel

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The following antibodies and staining reagents were used in this study: CD4 (RM4–5; BioLegend), CD8α (53–6.7; BioLegend), Thy1.1 (OX-7; BioLegend), CD44 (1M7; BioLegend), CD122 (5H4; BioLegend), PNA (FL-1071; Vector Laboratories Inc.), PSGL-1 (2PH1; BD Biosciences), CCR7 (4B12; BioLegend), CD43 Activation-Associated Glycoform (1B11; BioLegend), CD16/32 (2.4G2; Bio X Cell), Ly6C (HK1.4; BioLegend), Sca-1 (E13–161.7; BioLegend), CD34 (MED14.7; BioLegend), I-A^b (AF6–120.1; BioLegend), MHC Class I (34–1-25; Invitrogen), I-A/I-E (M5/114.15.2; BioLegend), CD119 (IFNγR a chain) (2E2; BioLegend), Goat anti-Human IgG Fc (Polyclonal; Invitrogen/eBioscience), eBioscience Streptavidin (Invitrogen), Rat IgG2a,

κ

Isotype Ctrl (RTK2758; BioLegend), Ghost Red 780 Viability Dye (Tonbo biosciences), CD45.2 (104; BioLegend), IFNγ (XMG1.2; BioLegend) and TNFα (MP6-XT22; BioLegend).

, & MacKenzie M.A. (2001). Standing watch. CMAJ: Canadian Medical Association Journal, 164(10), 1471.

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Profiling Tumor-Infiltrating T Cells

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For the analysis of T cells in the tumor in vivo, dissected tumors were digested by freshly prepared collagenase I (1 mg/mL, 17100–017, Thermo Fisher Scientific), collagenase IV (250 U/mL, 17100–019, Thermo Fisher Scientific), DNase (100 U/mL, Sigma-Aldrich, USA) solution for 60 min. Dissociated cells were filtered through a 40 μm mesh and separated by Ficoll gradient media (17–1440-02, GE Healthcare, USA) to obtain TILs. The isolated cells were then labeled for CD3 (145–2C11, BioLegend), CD4 (RM4–4, BioLegend), CD8 (53–6.7, eBioscience), CD45 (30-F11, BioLegend), PD-1 (29F.1.A12, BioLegend), FOXP3 (MF23, BD Bioscience), Interferon-γ (IFN-γ, XMG1.2, BioLegend) and Live/Dead Fixable Aqua Dead cell stain kit (L34957, Invitrogen) to identify and characterize T cell subsets in tumors. The fluorescence data were acquired on a Fortessa flow cytometer and analyzed with FlowJo software ver. 10.

Katagiri W., Yokomizo S., Ishizuka T., Yamashita K., Kopp T., Roessing M., Sato A., Iwasaki T., Sato H., Fukuda T., Monaco H., Manganiello S., Nomura S., Ng M.R., Feil S., Ogawa E., Fukumura D., Atochin D.N., Choi H.S, & Kashiwagi S. (2022). Dual near-infrared II laser modulates the cellular redox state of T cells and augments the efficacy of cancer immunotherapy. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(10), e22521.

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Multiparameter Flow Cytometry Analysis

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Whole spinal cord infiltrated cells or 2.0×10⁵ lymphocytes were stained with 2
µg/ml monoclonal antibodies (mAbs) after Fcγ receptor blocking with
2.4G2 Ab. After cell surface staining, cells were fixed with 4% paraformaldehyde and
permeabilized with 0.1% saponin, and then intracellular staining was performed. Anti-mouse
CD11c (N418 or HL3), CD4 (RM4-5 or GK1.5), CD8 (53-6.7), CD40 (3/2, 3), CD80 (16-10A1),
CD86 (GL-1), major histocompatibility complex class II (MHC class II) (M5/114.15.2),
granulocyte-differentiation antigen-1 (Gr-1) (RB6-8C5), IFN-γ (XMG1.2) and IL-17A
(TC11-18H10.1) were purchased from Biolegend (USA). Phcoerythrin (PE)-conjugated IL-17A
(TC11-18H10) and biotin-conjugated anti-mouse OX40 ligand (OX40L) (RM134L) were purchased
from BD Pharmingen (USA). PE/Cy7-conjugated streptavidin was purchased from BD Bioscience
(USA). Stained cells were detected with a FACSCanto II Flow Cytometer (Becton, Dickinson
and Company, USA) and analyzed with BD FACS Diva (Becton, Dickinson and Company) and
FlowJo software (Tree Star, USA). Dead cells which stained by 7-Amino Actinomycin D
(7-AAD) were excluded.

SENO A., MARUHASHI T., KAIFU T., YABE R., FUJIKADO N., MA G., IKARASHI T., KAKUTA S, & IWAKURA Y. (2014). Exacerbation of experimental autoimmune encephalomyelitis in mice deficient for DCIR, an inhibitory C-type lectin receptor. Experimental Animals, 64(2), 109-119.

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Tumor Digestion and Single-Cell Analysis

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Tumors were digested into single-cell suspensions as follows: Tumors were cut into small pieces using a scalpel and needle, and incubated in 40 mL digest media comprising RPMI-1640 with 2 mg/mL collagenase A (Roche, Cat. No. 11088793001) and 40units/mL DNase-I (Sigma-Aldrich, Cat. No. D5025) at 37°C with agitation for 1 hour. Tumor digests were then filtered through a 70 μm cell strainer (BD Falcon) and resuspended in FACS buffer (PBS + 2% FBS + 1 mM EDTA) for staining. Spleens were mashed through a 70 μm cell strainer and resuspended in FACS buffer for staining. Antibodies included CD3 (clone 145–2 C11), CD4 (RM4-R), CD8 (53–6.7), CD44 (IM7), CD62L (MEL-14), TIM-3 (B8.2 C12), LAG-3 (C9B7W), PD-1 (29 F.1A12), IFN-γ (XMG1.2), and TNF (MP6-XT22), all from BioLegend (San Diego, CA). Samples were acquired on an Accuri C6 Flow Cytometer (BD Biosciences) and analyzed with the FlowJo software (version 10.4). For cytokine assessment, digested cells were plated at 10⁶ cells/mL in a 96 well round bottom plate with Cell Activation Cocktail (BioLegend) overnight prior to intracellular staining, as recommended by the manufacturer using Fixation Buffer (BioLegend) and Intracellular Staining Permeabilization Wash Buffer 10X (BioLegend). Gating strategy is shown in Supplementary Figure S6.

White M.G., Szczepaniak Sloane R., Witt R.G., Reuben A., Gaudreau P.O., Andrews M.C., Feng N., Johnson S., Class C.A., Bristow C., Wani K., Hudgens C., Nezi L., Manzo T., De Macedo M.P., Hu J., Davis R., Jiang H., Prieto P., Burton E., Hwu P., Tawbi H., Gershenwald J., Lazar A.J., Tetzlaff M.T., Overwijk W., Woodman S.E., Cooper Z.A., Marszalek J.R., Davies M.A., Heffernan T.P, & Wargo J.A. (2021). Short-term treatment with multi-drug regimens combining BRAF/MEK-targeted therapy and immunotherapy results in durable responses in Braf-mutated melanoma. Oncoimmunology, 10(1), 1992880.

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Comprehensive Flow Cytometry Analysis

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Flow cytometry was performed using a FACSaria II (Becton Dickinson) and analyzed with FlowJo software (Tree Star). Fluorochrome-conjugated MAbs specific for: CD45 (clone 30-F11), CD45.1 (A20), CD11b (M1/70), CD3 (17A2), CD8α (53-6.7) CD4 (RM4-5), CD19 (1D3/CD19), CD11c (N418), CD26 (H194-112), CD88 (20/70), CD172α (P84), PDCA1 (927), Ly6c (AL-21), F4/80 (MB8), Ly6g (1A8), MHC-II (M5/114.15.2), PD-1 (29F.1A12), PD-L1 (10F.9G2), PD-L2 (TY25), Caspase 3, and Annexin V were purchased either from BD Biosciences, R&D, Biolegend, Santa Cruz, or Abcam.
For intracellular staining, cells were stimulated for 4 h with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml, Sigma-Aldrich) and ionomycin (500 ng/ml, Sigma-Aldrich) in the presence of GolgiPlug (1:1,000, BD Pharmigen), permeabilized using a Cytofix/Cytoperm Plus kit (BD Bioscience) and stained with the following fluochrome-conjugated MAbs: GM-CSF (MP1-22E9), IL-17A (TC11-18H10.1), and IFN-γ (XMG1.2) from Biolegend and BD Pharmingen. Dead cells were excluded using L/D stain (BD Pharmingen). Data were acquired on a FACSAria Fusion (BD Biosciences) and analyzed using FlowJo software (TreeStar).

Casella G., Rasouli J., Thome R., Descamps H.C., Vattikonda A., Ishikawa L., Boehm A., Hwang D., Zhang W., Xiao D., Park J., Zhang G.X., Alvarez J.I., Rostami A, & Ciric B. (2020). Interferon-γ/Interleukin-27 Axis Induces Programmed Death Ligand 1 Expression in Monocyte-Derived Dendritic Cells and Restores Immune Tolerance in Central Nervous System Autoimmunity. Frontiers in Immunology, 11, 576752.

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Comprehensive Immune Cell Phenotyping

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Antibodies against CD4 (RM4-5 or GK1.5), CD8 (53-6.7), CD45R/B220 (RA3-6B2), CD45.2 (104), TER-119 (TER-119), CD31 (390), gp38 (Podoplanin; 8.1.1), CD3 (17A2), Klrg1 (2F1), CD127 (A7R34), CD11c (N418), CD11b (M1/70), CD19 (6D5), NK1.1 (PK136), CD90.2 (30-H12), GR-1 (RB6-8C5), IFN-γ (XMG1.2), TNF (MP6-XT22) and IL-2 (JES6-5H4) were from Biolegend, Pharmingen and eBioscience. All fluorescently labelled monoclonal antibodies were used at a 1:100 dilution, except for gp38 (Podoplanin) and TER-119, which were diluted 1:1,000, and 1:10, respectively. Dead cells were excluded with Zombie UV Fixable Viability Kit (Biolegend, cat. no. 423108). OVA- (SIINFEKL) and P1A epitope- (LPYLGWLVF) specific CTLs were identified as peptide-MHC class I tetramers (TCMetrix) binding cells amongst CD8⁺B220⁻ lymphocytes. BD Trucount Absolute Counting Tubes were used to determine absolute cell counts in blood. Spleen lymphocyte counts
were determined in a Neubauer chamber. Peptide-MHC tetramer-binding CTLs were back calculated. Cytokine profiles after restimulation with peptide (SIINFEKL for OVA, TYLPANASL for Her2; ProImmune) were determined in intracellular cytokine assays11 (link). Samples were measured on BD LSRFortessa and Beckman Coulter Gallios flow cytometers and were analysed using FlowJo software (Tree Star, Ashland, OR).

Kallert S.M., Darbre S., Bonilla W.V., Kreutzfeldt M., Page N., Müller P., Kreuzaler M., Lu M., Favre S., Kreppel F., Löhning M., Luther S.A., Zippelius A., Merkler D, & Pinschewer D.D. (2017). Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy. Nature Communications, 8, 15327.

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Isolation and Characterization of Ascophyllan

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Ascophyllan was purified from A. nodosum as a sulfated fucan preparation [13 (link),47 (link)]. Briefly, in the first step, a hot water extraction was performed to obtain the total soluble polysaccharide fraction, which contained ascophyllan, fucoidan, and trace levels of alginate. In the second step, alginate was removed by alginate lyase digestion. The third step was the selective precipitation of ascophyllan in the presence of NaCl and alcohol. In this step, ascophyllan was separated from fucoidan. Finally, a composition analysis was performed to characterize the isolated polysaccharides [45 (link)]. Fucoidan from M. pyrifera was obtained from Sigma Aldrich (St. Louis, MO, USA). Fluorescence-conjugated Abs against IgG1, IgG2a, CD3 (17A2), CD69 (H1.2F3), NK1.1 (PK136), Ki-67 (16A8), and IFN-γ (XMG1.2) were purchased from BioLegend (San Diego, CA, USA).

Zhang W., Okimura T., Oda T, & Jin J.O. (2019). Ascophyllan Induces Activation of Natural Killer Cells in Mice In Vivo and In Vitro. Marine Drugs, 17(4), 197.

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