Rhifn γ

Manufactured by R&D Systems

Sourced in United States

RhIFN-γ is a recombinant human interferon-gamma protein. Interferon-gamma is a cytokine that plays a crucial role in the immune system's response to pathogens.

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Lab products found in correlation

Rhil 4, by R&D Systems (4 mentions) Rhil 13, by R&D Systems (4 mentions) Rhil 15, by R&D Systems (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Rhgm csf, by R&D Systems (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Gm csf, by R&D Systems (2 mentions) Lymphoprep, by STEMCELL (2 mentions) Penicillin streptomycin, by Lonza (2 mentions) Rhil 10, by R&D Systems (2 mentions) Rhtnf α, by R&D Systems (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions) Ifn γ, by R&D Systems (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Il 15, by R&D Systems (1 mentions) Rhtnf, by R&D Systems (1 mentions) Rhil 17a, by R&D Systems (1 mentions) Rhil 22, by R&D Systems (1 mentions) Nunc maxisorp, by Thermo Fisher Scientific (1 mentions)

19 protocols using rhifn γ

Monocyte Polarization to TAM and M1

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CD14⁺ monocytes were plated in serum-free X-VIVO^™ 15 medium (Lonza) with either 100 ng/mL recombinant human macrophage colony-stimulating factor (M-CSF, Immunotools) or 100 ng/mL recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF, Miltenyi Biotec) to differentiate the monocytes toward TAM or M1, respectively. On day 4, half-medium change was performed. On day 7, the cells were polarized toward TAM or M1 for 48 h as follows: To obtain TAM, 100 ng/mL M-CSF, 50 mM lactate (Sigma-Aldrich), 200 ng/mL prostaglandin E2 (PGE2; Sigma-Aldrich) were added together with either L363 cells in a 1:30 L363:monocytes ratio or with L363 supernatant. To obtain M1 macrophages, 100 ng/mL GM-CSF, 20 ng/mL recombinant human IFN-γ (rhIFN-γ, R&D Systems) and 10 ng/mL LPS (Sigma-Aldrich) were added to the medium.
To some conditions, supernatant from IL-2-activated NK cells or rhIFN-γ (R&D Systems) was added to the polarized macrophages for 24 h, referred to as NK supernatant. Additionally, 5 µg/mL LEAF purified anti-human CD119/IFN-γ R alpha chain, mouse IgG1 κ blocking antibody (clone GIR-208) or 5 μg/mL LEAF purified mouse IgG1 κ isotype control (MGI-45/ MOPC-21, both Biolegend) was added 30 min prior to adding the NK supernatant.

Ehlers F.A., Mahaweni N.M., van de Waterweg Berends A., Saya T., Bos G.M, & Wieten L. (2023). Exploring the potential of combining IL-2-activated NK cells with an anti-PDL1 monoclonal antibody to target multiple myeloma-associated macrophages. Cancer Immunology, Immunotherapy, 72(6), 1789-1801.

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Immune Activation Reagent Protocol

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Blocking antibodies against human CD40L, IFN-g, IL-15 and rhIL-15 and rhIFN-g were purchased from R&D systems (Minneapolis, MN, USA). DAPI, CFSE, and CellMask deep red plasma membrane stain purchased from Thermo Fisher Scientific (USA). All fluorescent antibodies and isotype control IgG were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Wang H., Jiang H., Teles R., Chen Y., Wu A., Lu J., Chen Z., Ma F., Pellegrini M, & Modlin R.L. (2020). Cellular, molecular and immunological characteristics of Langhans multinucleated giant cells programmed by IL-15. The Journal of investigative dermatology, 140(9), 1824-1836.e7.

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Keratinocyte Cytokine Response Assay

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Keratinocytes were isolated from skin biopsy specimens, as previously described. 21 Ten thousand keratinocytes at passage 3 or 4 were plated in Abbreviations used HBD2: Human b-defensin 2 MX1: Myxovirus 1 NLP: Never-lesional skin from patients with psoriasis pDC: Plasmacytoid dendritic cell rh: Recombinant human 48-well plates. At 100% confluence, cultures were exposed to recombinant human (rh) IL-17A (5 ng/mL), rhIL-22 (5 ng/mL), rhTNF (5 ng/mL), rhIFN-a (5 U/mL), or rhIFN-g (5 ng/mL) (all from R&D Systems, Minneapolis, Minn) for 16 hours.

Gallais Sérézal I., Hoffer E., Ignatov B., Martini E., Zitti B., Ehrström M, & Eidsmo L. (2019). A skewed pool of resident T cells triggers psoriasis-associated tissue responses in never-lesional skin from patients with psoriasis. The Journal of allergy and clinical immunology, 143(4).

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Antiviral Efficacy of Ribavirin and IFNs

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Ribavirin, provided by Yamasa-Shouyu Co., Ltd. (Choshi, Japan), was dissolved in phosphate-buffered saline at a concentration of 100 mg/ml. IFNα (universal Type I IFN, PBL InterferonSource), β (human IFN beta 1a, PBL InterferonSource), and γ (rhIFNγ, R & D Systems) were dissolved in DMEM supplemented with 2 % FCS at 500,000 U/ml, 500,000 U/ml, and 200 μg/ml, respectively. For treatment of cells, these drugs were further diluted with cell culture medium at indicated concentration (s), alone or in indicated combinations. Cells were pre-treated with drugs at indicated concentrations for 1 h. One hundred TCID₅₀ of SFTS virus was added to the culture without removal of the drugs, and then cultured for 3 days. Culture supernatants were harvested and subjected to virus titration [14 (link)].

Shimojima M., Fukushi S., Tani H., Taniguchi S., Fukuma A, & Saijo M. (2015). Combination effects of ribavirin and interferons on severe fever with thrombocytopenia syndrome virus infection. Virology Journal, 12, 181.

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Enzyme-Linked Immunosorbent Assay for Anti-Cytokine Autoantibodies

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Briefly, 96-well plates (Nunc Maxisorp, Thermo Fisher Scientific) were coated by incubation overnight at 4°C with 1 µg/mL rhGM-CSF or rhIFN-γ (R&D Systems). The plates were washed in PBS 0.005% Tween, blocked with 1 × PBS supplemented with 5% nonfat milk powder, washed, and incubated for 2 h at room temperature with 1:50, 1:250, and 1:1,000 dilutions of plasma from patients and healthy controls, or plasma from a patient with cryptococcal meningitis and a PAP patient previously shown to have high titers of anti-GM-CSF auto-Abs, as a positive control. The plates were washed and horseradish peroxidase (HRP)–conjugated Fc-specific IgG (polyclonal goat antiserum against human IgG, Nordic Immunological Laboratories) was added to a final concentration of 1 µg/mL. Plates were incubated for 1 h at room temperature and washed. Substrate was added, and optical density was measured. The antibody specificity controls were plasma samples from a patient with high titers of auto-Ab against IFN-γ, and a patient with APS-1 and high titers of auto-Ab against IL-17A, IL-17F, IL-22, IFN-α, and IFN-ω.

Arango-Franco C.A., Migaud M., Ramírez-Sánchez I.C., Arango-Bustamante K., Moncada-Vélez M., Rojas J., Gervais A., Patiño-Giraldo S., Perez-Zapata L.J., Álvarez Álvarez J.A., Orrego J.C., Roncancio-Villamil G., Boisson-Dupuis S., Jouanguy E., Abel L., Casanova J.L., Bustamante J., Arias A.A., Franco J.L, & Puel A. (2023). Anti-GM-CSF Neutralizing Autoantibodies in Colombian Patients with Disseminated Cryptococcosis. Journal of Clinical Immunology, 43(5), 921-932.

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Evaluating Skin Responses to Cytokine Stimuli

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Full-thickness human skin models (MatTek Corp., Ashland, MA, U.S.A.) (n = 4) were incubated in assay media (MatTek Corp.) supplemented with or without rh-IL-17 (R&D Systems, Minneapolis, MN, U.S.A.) 200 ng mL⁻¹, rh-IL-22 (Peprotech Inc., Rocky Hill, NJ, U.S.A.) 200 ng mL⁻¹ 200, or rh-IFN-γ (R&D Systems, Minneapolis, MN, U.S.A.) 20 ng mL⁻¹, for 2 days. On day 2, the skin models were harvested for microarray analyses. The same concentrations used for treating in vitro monolayer keratinocytes were applied for RHE, as they were proved effective in gene modulation as previously described by our group [12] (link).

Chiricozzi A., Nograles K.E., Johnson-Huang L.M., Fuentes-Duculan J., Cardinale I., Bonifacio K.M., Gulati N., Mitsui H., Guttman-Yassky E., Suárez-Fariñas M, & Krueger J.G. (2014). IL-17 Induces an Expanded Range of Downstream Genes in Reconstituted Human Epidermis Model. PLoS ONE, 9(2), e90284.

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Expansion of Human CIK Cells

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CIK cells were obtained from healthy donors as previously described.29 (link) Briefly, PBMCs from buffy coats provided from the Blood Bank of Padua Hospital, Italy, were isolated by density gradient centrifugation (Lymphoprep, STEMCELL Technologies Inc, BC, Canada). Cells were resuspended in Roswell Park Memorial Institute (RPMI) 1640 (Euroclone, Milan, IT) with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Fisher, Massachusetts, USA), 1% ultraglutamine, 1% HEPES buffer and 1% penicillin/streptomycin (Lonza, Switzerland) (complete RPMI) and incubated at 37°C, 5% CO₂. At day 0, 1000 U/mL of recombinant human interferon-γ (rhIFN-γ) (R&D, MN, USA) was added to the medium, followed 24 hours later by 50 ng/mL of anti-CD3 pure human-functional grade antibody (OKT-3, Miltenyi Biotec, California, USA) and recombinant human interleukin 2 (rhIL-2) (Proleukin, Novartis, Switzerland) at 500 IU/mL. Fresh rhIL-2 was added to the medium every 3–4 days.

Pietà A.D., Cappuzzello E., Palmerini P., Ventura A., Visentin A., Astori G., Chieregato K., Mozzo V., Perbellini O., Tisi M.C., Trentin L., Visco C., Ruggeri M., Sommaggio R, & Rosato A. (2021). Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells. Journal for Immunotherapy of Cancer, 9(7), e002475.

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Macrophage Kynureninase Silencing Assay

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Macrophages were stimulated with recombinant human interferon-γ (rhIFN-γ; R＆D Systems, Minneapolis, MN, USA) at a final concentration of 10 ng/mL in serum- and antibiotic-free RPMI1640 for 24 h. Macrophages were transfected by incubating human kynureninase siRNA (Silencer® Select, siRNA ID; s17103 and/or s17104; Life Technologies, Carlsbad, CA, USA) or negative control siRNA (Silencer® Select, Negative Control #2 siRNA; Life Technologies) at a final concentration of 37.5 nM under the serum- and antibiotic-free RPMI1640 for 24 h, using Viromer® Blue transfection reagent (Lipocalyx). To examine siRNA-induced cell damage, lactate dehydrogenase (LDH) in the medium was measured using the LDH cytotoxicity assay kit (Nacalai Tesque, Kyoto, Japan). First, 100 µL of the supernatant from each well in which macrophages had been cultured under the indicated conditions was transferred to an optically transparent 96-well plate. A total of 100 µL of substrate solution was added and incubated for 20 min at RT under protection from light to each well of the above plate. Finally, 50 µL of stop solution was added to each well, and the absorbance was measured at 490 nm using a microplate reader. The results are presented as absorbance values and expressed as mean±standard deviation (SD).

Nishimura M., Yamashita A., Matsuura Y., Okutsu J., Fukahori A., Hirata T., Nishizawa T., Ishii H., Maekawa K., Nakamura E., Kitamura K., Nakamura K, & Asada Y. (2020). Upregulated Kynurenine Pathway Enzymes in Aortic Atherosclerotic Aneurysm: Macrophage Kynureninase Downregulates Inflammation. Journal of Atherosclerosis and Thrombosis, 28(11), 1214-1240.

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Cytokine-Induced Killer Cell Generation

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CIK cells were isolated from PBMCs derived from healthy blood donors and generated according to the established CIK cell generation standard protocol (20). CIK cells were grown in RPMI 1640 medium with 2.5% (v/v) of HEPES buffer (both from PAN-Biotech GmbH, Aidenbach, Germany), 10% (v/v) heat inactivated FCS, 1% P/S (both Gibco^® by life science technologies™, Carlsbad, CA, U.S.A.) and were incubated aseptically at 37 °C with 5% CO₂. The flask incubated at 37 °C with 5% CO₂ for 1–2 h to get rid of monocytes that adhere to the flask. Of RhIFNγ 1000 IU/mL (R&D Systems Inc., Minneapolis, ME, U.S.A.) was initially added to CIK cells for 24 h, followed by 300 IU/mL interleukin-2 (Novartis Pharma AG, Basel, Switzerland), 50 ng/mL anti-human anti-CD3 mAb and 100 IU/mL interleukin-1β (both from eBioscience Inc., San Diego, CA, U.S.A.). interleukin-2 was replenished every third day of culture. The cells were used for investigation after eight days of culture.

Dehno M.N., Li Y., Weiher H, & Schmidt-Wolf I.G. (2020). Increase in Efficacy of Checkpoint Inhibition by Cytokine-Induced-Killer Cells as a Combination Immunotherapy for Renal Cancer. International Journal of Molecular Sciences, 21(9), 3078.

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Isolation and Differentiation of Monocyte-Derived Macrophages

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Peripheral blood mononuclear cells (PBMCs) were isolated from Leukocyte Reduction System chambers (Bloodworks Northwest) with a Lymphoprep (Stemcell Technologies) density gradient applied to SepMate-50 tubes (Stemcell Technologies) and cryopreserved in FBS/10% dimethylsulfoxide (DMSO; Sigma). CD14⁺ monocyte isolation from cryopreserved PBMCs was performed using EasySep Human CD14 Positive Selection Kits (Stemcell Technologies), according to the manufacturer’s instructions. With minor variations in timing, macrophages were generated by culturing CD14⁺ monocytes in RP10 with 10 ng/mL recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems) for 6 days at 37°C, 5% CO₂, refreshing cytokine on day 3. Macrophages were detached using 0.25% Trypsin-EDTA (Gibco) and cell scrapers, and replated in RP10. For transduction, lentivirus was added to culture and macrophages harvested for downstream assays 5–7 days later, replacing media every 2–4 days. For downstream assays, macrophages were detached using 0.25% Trypsin-EDTA, TrypLE, TrypLE Select or Versene (Gibco) and cell scrapers. To test IL-12 receptor (IL-12R) expression, macrophages were cultured in 100 ng/mL lipopolysaccharide (LPS) (Sigma) and 20 ng/mL each rhIFNγ, rhIL-4, rhIL-10 and rh transforming growth factor beta 1 (TGF-β1) (all R&D).

Brempelis K.J., Cowan C.M., Kreuser S.A., Labadie K.P., Prieskorn B.M., Lieberman N.A., Ene C.I., Moyes K.W., Chinn H., DeGolier K.R., Matsumoto L.R., Daniel S.K., Yokoyama J.K., Davis A.D., Hoglund V.J., Smythe K.S., Balcaitis S.D., Jensen M.C., Ellenbogen R.G., Campbell J.S., Pierce R.H., Holland E.C., Pillarisetty V.G, & Crane C.A. (2020). Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses. Journal for Immunotherapy of Cancer, 8(2), e001356.

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