Ifn γ

Manufactured by Fujifilm

Sourced in Japan, United States

IFN-γ is a recombinant human interferon-gamma protein used in laboratory research. It is a cytokine that plays a key role in immune system regulation and functions.

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Lab products found in correlation

Lipopolysaccharides (lps), by Merck Group (5 mentions) Tnf α, by Fujifilm (3 mentions) Il 10, by Fujifilm (3 mentions) Ifn β, by Thermo Fisher Scientific (2 mentions) Interleukin 4 (il 4), by R&D Systems (2 mentions) Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Il 1β, by Fujifilm (2 mentions) Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Polyinosinic polycytidylic acid poly 1 c, by Merck Group (1 mentions) T75 flask, by Corning (1 mentions) Interleukin 4 (il 4), by Gentaur (1 mentions) Granulocyte macrophage colony stimulating factor, by Gentaur (1 mentions) Transit x2, by Mirus Bio (1 mentions) Gdc 0994, by Selleck Chemicals (1 mentions) Rpmi 1640, by Lonza (1 mentions) 96 well round bottom plate, by Corning (1 mentions) Il 27, by R&D Systems (1 mentions)

19 protocols using ifn γ

Interferons and Innate Immune Stimulation

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Recombinant human IFN-α2, IFN-β, IFN-λ3 (PBL
Interferon Source) and IFN-γ (Shenandoah Biotechnology) were used at
25–100 ng/ml (IFN-α2), 25–500 IU/ml (IFN-β), 100
ng/ml IFN-λ3 or 5 ng/ml (IFN-γ), respectively. For RIG-I
stimulations, poly U/UC RNA was synthesized by in vitrotranscription as previously described⁴⁰. Poly U/UC RNA was transfected at 0.2–1
μg/ml using TransIT-X2 (Mirus Bio) according to the
manufacturer’s instructions. Stimulations of TLR3 and TLR7/8 were
performed using 10 μg/ml floating poly(I:C) or 1 μg/ml R848 (both
InvivoGen) in culture media.

Schwerk J., Soveg F.W., Ryan A.P., Thomas K.R., Hatfield L.D., Ozarkar S., Forero A., Kell A.M., Roby J.A., So L., Hyde J.L., Gale M J.r., Daugherty M.D, & Savan R. (2019). RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions. Nature immunology, 20(12), 1610-1620.

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Monocyte-Derived Dendritic Cell Protocol

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PBMCs were incubated in T-75 flasks (Corning) for 2 hours and nonadherent cells (mostly peripheral blood lymphocytes) were saved. Adherent cells (mostly monocytes) were washed three times with phosphate-buffered saline, differentiated over 6 days into immature DCs with granulocyte-macrophage colony-stimulating factor (Gentaur, Brussels, Belgium, 50 ng/ml) and IL-4 (Gentaur, 25 ng/ml). Medium was changed every 2 days. Immature DCs were then maturated for 24 hours in culture medium with IFN-γ (Shenandoah Biotechnology, Warwick, PA, 1,000 IU/ml), polyinosinic:polycytidylic acid (poly(I:C); Sigma-Aldrich, St Louis, MO, 20 µg/ml) and R848 (InvivoGen, San Diego, CA, 2.5 μg/ml).

Jin C., Yu D., Hillerdal V., Wallgren A., Karlsson-Parra A, & Essand M. (2014). Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors. Molecular Therapy. Methods & Clinical Development, 1, 14001-.

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Differentiation and Activation of Murine BMDMs

Cited 1 time

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Bone marrow-derived macrophages (BMDMs) from single-cell suspensions of tibia and femur marrow were differentiated in vitro as previously described [21 (link)]. Briefly, murine bone marrow was cultured in RPMI 1640 (Lonza, Walkersville, MD) supplemented with 30% L-cell conditioned media, 20% Fetal Calf Serum and penicillin/streptomycin for a period of six days. Adherent BMDMs were harvested and replated in minimal media for a rest phase of 12–18 hours. Following this rest phase, BMDMs were pre-treated in certain conditions with CCL2 (R&D Systems, Minneapolis, MD) for a period of 12–18 hours, were indicated. Stimulations with IFNγ (Shenandoah Biotechnology, Warwick, PA), IL-4 (Shenandoah), and LPS (0111:B4, Sigma, St. Louis, MO) were performed following the rest and pretreatment phase. Reported endotoxin levels in the utilized recombinant cytokines are as follows: <0.01 EU/μg for CCL2 and <1 EU/μg for IFNγ and IL-4. The ERK1/2 inhibitor GDC-0994 (Selleck Chemicals, Houston, TX) was resuspended in DMSO and used at a final concentration of 50 nM in cell culture assays.

Carson WF I.V., Salter-Green S.E., Scola M.M., Joshi A., Gallagher K.A, & Kunkel S.L. (2017). Enhancement of macrophage inflammatory responses by CCL2 is correlated with increased miR-9 expression and downregulation of the ERK1/2 phosphatase Dusp6. Cellular immunology, 314, 63-72.

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Quantifying MHC-I Expression in B16-OVA Cells

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Following exposure to either γ-irradiation or UV-irradiation or treatment with 50 ng/mL of interferon-γ (IFN-γ) (Shenandoah Biotechnology, USA), B16-OVA cells were incubated for 18 hrs. in a 6-well plate at a density of 1.0x10⁶ cells/well. After incubation, the cells were harvested and washed with 1X PBS, then transferred to a 96-well round-bottom plate (Corning, USA). Cells were then washed twice in flow staining buffer (1X PBS, 0.1% sodium azide, 1% BSA) and then stained with PE-anti-MHC-I (Biolegend, clone: 28-8-6) for 30 min at 4°C. Data was acquired using a CytoFLEX flow cytometer and analyzed using FlowJo software (BD, USA).

Seaver K., Kourko O., Gee K., Greer P.A, & Basta S. (2022). IL-27 Improves Prophylactic Protection Provided by a Dead Tumor Cell Vaccine in a Mouse Melanoma Model. Frontiers in Immunology, 13, 884827.

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Differentiation of Bone Marrow Cells into Immune Cells

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Bone marrow cells from indicated animals were isolated and cultured in L-cell conditioned media (RPMI 1640, 20% v/v Fetal Bovine Serum/FBS, 30% v/v L929 conditioned supernatant) for a period of seven days. At the end of the differentiation culture, adherent cells were harvested, replated and cultured in RPMI 1640 with 10% v/v FBS and penicillin/streptomycin. The concentrations of the indicated stimuli were as follows: IFNγ (Shenandoah Biotechnology, Warwick, PA), IL-4 (Shenandoah), IL-12 (Shenandoah), IL-23 (R&D Systems, Minneapolis, MN), IL-27 (R&D), IL-28β (R&D), IFNα3 (PeproTech, Rocky Hill, NJ), IFNβ (PeproTech) = all at 10 ng/ml. LPS (Sigma Aldrich, St. Louis, MO) = 100 ng/ml. CpG & pI:C (InvivoGen, San Diego, CA) = 10 μg/ml.

Carson WF I.V., Cavassani K.A., Soares E.M., Hirai S., Kittan N.A., Schaller M.A., Scola M.M., Joshi A., Matsukawa A., Aronoff D.M., Johnson C.N., Dou Y., Gallagher K.A, & Kunkel S.L. (2017). The STAT4/MLL1 epigenetic axis regulates the antimicrobial functions of murine macrophages. Journal of immunology (Baltimore, Md. : 1950), 199(5), 1865-1874.

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Bone marrow and spleen macrophage generation

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Bone marrow-MΦ and Sp-MΦ were generated as previously described (8 (link), 23 (link), 51 (link)). Briefly, cells (for both BM-MΦ and Sp-MΦ) were cultured in 6 well plates (Corning) with conditioned RPMI 1640 (Gibco) medium (CM) supplemented with 10% fetal calf serum (Fisher Scientific), M-CSF media and 50 µg/mL gentamycin. On days 3 and 5 of culturing, non-adherent cells were removed and fresh CM was added. Sp-MΦ and BM-MΦ were cultured for 6–7 days in total before activation and testing for their functions as described below. For M(LPS + IFN-γ) MΦ: cells were primed with IFN-γ (25 ng/mL for 16–18 h; Shenandoah Biotechnology) followed by Escherichia coli LPS (O55:B5, 100 ng/mL for 6 h; Sigma-Aldrich). To induce M(IL-4) MΦ, polarization cells were treated with rIL-4 (20 ng/mL for 18–24 h; Shenandoah Biotechnology). Unstimulated control MΦ (M0) were placed in RPMI 10% FCS for 18–24 h.

Mulder R., Banete A., Seaver K, & Basta S. (2017). M(IL-4) Tissue Macrophages Support Efficient Interferon-Gamma Production in Antigen-Specific CD8+ T Cells with Reduced Proliferative Capacity. Frontiers in Immunology, 8, 1629.

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Impact of IFN-γ on TNBC Cell Behavior

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To study the effect of IFN-γ on TNBC cells, EpRas cells were plated in 10-cm dishes. After 24 h, when the cells became 70–80% confluent, either a sterile water control or IFN-γ (10 ng ml⁻¹; catalogue number 100-77; Shenandoah Biotechnology) was added directly to the plates with 10 ml fresh media. The cells were cultured further for 48 h and harvested for qPCR, western blotting, FACS or tumour cell injections. For injection into the mice, cells were trypsinized, washed with phosphate-buffered saline (PBS) and counted for MFP or intravenous injection into the mice.

Singh S., Kumar S., Srivastava R.K., Nandi A., Thacker G., Murali H., Kim S., Baldeon M., Tobias J., Blanco M.A., Saffie R., Zaidi M.R., Sinha S., Busino L., Fuchs S.Y, & Chakrabarti R. (2020). Loss of ELF5–FBXW7 stabilizes IFNGR1 to promote the growth and metastasis of triple-negative breast cancer through interferon-γ signalling. Nature cell biology, 22(5), 591-602.

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Immunotherapy Combination Dosing Protocol

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Dosing with pretreatment drugs commenced on day 15 for AB1 and Renca, day 10 for AE17 or day 8 for B16. IFNγ (Shenandoah Biotechnology) was dosed s.c. into tumor area at 50,000 units daily for 3 days. Poly(I:C) (HMW, Invivogen) was dosed s.c. into tumor area at 50 μg daily for 3 days. The anti-IL-10 hybridoma (clone JES5.2A5) was cultured in IMDM containing 1% of FCS and gentamycin. Clarified supernatants were used to purify the antibody using affinity chromatography. The antibody was sterile formulated in PBS. Anti-IL-10 was dosed i.p. at 0.5 mg/mouse daily for 3 days. ICB was dosed 2 days after pretreatment drug schedule; day 20 for AB1 and Renca, day 15 for AE17 or day 13 for B16; for the ICB only group, dosing began at the same time as the pretreatment drug dosing commenced in the other arms.

Zemek R.M., De Jong E., Chin W.L., Schuster I.S., Fear V.S., Casey T.H., Forbes C., Dart S.J., Leslie C., Zaitouny A., Small M., Boon L., Forrest A.R.R., Muiri D.O., Degli-Esposti M.A., Millward M.J., Nowak A.K., Lassmann T., Bosco A., Lake R.A, & Lesterhuis W.J. (2019). Sensitization to immune checkpoint blockade through activation of a STAT1/NK axis in the tumor microenvironment. Science translational medicine, 11(501).

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Cytokine Array Analysis Protocol

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A cytokine array analysis (R&D system, Cat# ARY005) was performed according to the manufacturer's instructions. The concentration of each cytokine for stimulation was as follows: IFN-γ (Wako), 20 ng/ml; TNF-α (Wako), 20 ng/ml and IL-10 (Wako), 50 ng/ml [40 (link)]. The experiments for each stimulation were repeated at least twice, and similar results were obtained.

Sato M., Kawana K., Adachi K., Fujimoto A., Yoshida M., Nakamura H., Nishida H., Inoue T., Taguchi A., Ogishima J., Eguchi S., Yamashita A., Tomio K., Wada-Hiraike O., Oda K., Nagamatsu T., Osuga Y, & Fujii T. (2017). Regeneration of cervical reserve cell-like cells from human induced pluripotent stem cells (iPSCs): A new approach to finding targets for cervical cancer stem cell treatment. Oncotarget, 8(25), 40935-40945.

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Immunofluorescence Staining Reagents and Compounds

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Mouse monoclonal anti-GAPDH (Cat. No. M171-3), anti-EEA1 (Cat. No. M176-3), and anti-KDEL (Cat. No. M181-3) antibodies were from Medical and Biological Laboratories (Nagoya, Japan). Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 555-conjugated goat anti-mouse, anti-rat and anti-rabbit IgG were from ThermoFisher Scientific (Rockford, IL, USA). Rat monoclonal anti-LAMP1 (Cat. No. 553792) and mouse monoclonal anti-GM130 (Cat. No. 610823) were from BD Biosciences (Franklin Lakes, NJ, USA). Dexamethasone, etoposide, and IFN-γ were purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan); and rapamycin was from LC Laboratories (Boston, MA, USA). Other reagents, including the protease inhibitor cocktail and LPS, were purchased from Sigma Aldrich (Tokyo, Japan).

Tokuhisa M., Kadowaki T., Ogawa K., Yamaguchi Y., Kido M.A., Gao W., Umeda M, & Tsukuba T. (2020). Expression and localisation of Rab44 in immune-related cells change during cell differentiation and stimulation. Scientific Reports, 10, 10728.

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