Rhil 2

Manufactured by Novartis

Sourced in Switzerland, Japan, United States, Germany

RhIL-2 is a recombinant human interleukin-2 product. Interleukin-2 is a cytokine that plays a crucial role in the activation and proliferation of T cells. RhIL-2 is used as a research tool in various cell culture and immunological studies.

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Close spelling variants of this product

Recombinant human interleukin-2 (rhIL-2; (3 citations) Recombinant human IL-2 (rhIL-2; (2 citations)

47 protocols using rhil 2

Transduction and Activation of T Cells

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T cells derived from normal healthy donors were activated prior to transduction using 50 ng/mL anti-CD3 mAb (Miltenyi Biotec), 300 IU/mL rhIL-2 (rhIL-2; Novartis Pharmaceuticals), and 100 ng/mL rhIL-15 (NCI-Frederick) on day 0. T cells, Jurkat E6.1 cells, and CD8⁺ Jurkat E6.1 cells were transduced by spinoculation on day 3 as described elsewhere.10 (link), 31 (link) Transduced T cells or Jurkat E6.1 cells were purified by positive selection using anti-CD34 magnetic beads (Miltenyi Biotec) and maintained in complete T cell medium or Jurkat cell medium. The T cells were used in functional assays beginning on day 13.

Foley K.C., Spear T.T., Murray D.C., Nagato K., Garrett-Mayer E, & Nishimura M.I. (2017). HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer. Molecular Therapy Oncolytics, 5, 105-115.

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Isolation and Characterization of CIK Cells

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CIK cells were obtained from PBMCs of healthy donors isolated by means of Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation, according to standard protocols.^{10 (link)} PBMCs were plated in RPMI 1640 (Euroclone) supplemented with 10% heat-inactivated FBS (Gibco), 1% Ultraglutamine, 1% Hepes buffer, 1% penicillin/streptomycin (all from Lonza), at 37°C and 5% CO₂, and stimulated with rhIFN-γ (PeproTech) at 1000 U/ml at day 0. Twenty-four hours later, anti-CD3 mAb (OKT-3, Ortho Biotech Inc) at 50 ng/ml and rhIL-2 (Proleukin, Novartis) at 500 IU/ml were added to the culture medium; every 2–3 days medium was replenished and fresh rhIL-2 at 500 IU/ml was added. CIK cells phenotype was analyzed by multi-color flow cytometry, using the following antibodies: CD3-BV510 (clone UCHT1), IL-2-BV421 (clone 5344.111), CD25-APC (IL-2 Rα, clone M-A251), CD122-BV650 (IL-2Rβ, clone Mik-β3), CD132-BV786 (IL-2Rγ, clone AG184), from BD Bioscience; CD56-PE (clone HCD56), CD16a-FITC (clone 3G8), from BioLegend. Flow cytometry analysis was performed on either LSRII or Celesta, using DIVA software (BD Bioscience). Data analyzes were performed using FlowJo software (Treestar).

Sommaggio R., Cappuzzello E., Dalla Pietà A., Tosi A., Palmerini P., Carpanese D., Nicolè L, & Rosato A. (2020). Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells. Oncoimmunology, 9(1), 1777046.

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Isolation and Expansion of NK Cells

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NK cells were derived from healthy donors. Approval was obtained from the ethical committee of IRCCS Ospedale Policlinico San Martino (39/2012) of Genova (Italy), and informed consent was provided according to the Declaration of Helsinki. NK cells were purified from peripheral blood using the RosetteSep™ NK Cell Enrichment Cocktail (StemCell Technologies, 15025). Those populations displaying more than 95% of CD56⁺CD3⁻CD14⁻ NK cells were selected. Polyclonal NK cell lines were obtained by culturing purified NK cells at appropriate dilutions on irradiated feeder cells in the presence of 100 U/mL rhIL-2 (Proleukin, Novartis) and 1,5 ng/mL phytohemagglutinin (PHA, Gibco Ltd, 10576-015) in round-bottomed 96-well microtiter plates. After 3/4 weeks of culture the expanded NK cells were used for NK cell stimulation experiments.

Gaggero S., Bruschi M., Petretto A., Parodi M., Zotto G.D., Lavarello C., Prato C., Santucci L., Barbuto A., Bottino C., Candiano G., Moretta A., Vitale M., Moretta L, & Cantoni C. (2018). Nidogen-1 is a novel extracellular ligand for the NKp44 activating receptor. Oncoimmunology, 7(9), e1470730.

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Cultivation and Maintenance of T Cells

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HLA-A∗0201-positive TAP-deficient T2 cells (ATCC) and CD4 blasts were cultured in RPMI 1640 Glutamax media (GIBCO) supplemented with 10% FBS (Biowest) and penicillin/streptomycin (BioConcept). Primary CD8+ T cells were cultured in RPMI 1640 Glutamax media (GIBCO) supplemented with 8% Humsn serum (Biowest), non-essential amino acids (GIBCO), 2-mercaptoethanol (GIBCO), sodium pyruvate (GIBCO), HEPES (GIBCO), penicillin/streptomycin (BioConcept) and 150 U/ml of rhIL2 (Novartis). All cells were maintained at 37°C under 5% CO₂ atmosphere.

Schmidt J., Smith A.R., Magnin M., Racle J., Devlin J.R., Bobisse S., Cesbron J., Bonnet V., Carmona S.J., Huber F., Ciriello G., Speiser D.E., Bassani-Sternberg M., Coukos G., Baker B.M., Harari A, & Gfeller D. (2021). Prediction of neo-epitope immunogenicity reveals TCR recognition determinants and provides insight into immunoediting. Cell Reports Medicine, 2(2), 100194.

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Expansion of Pmel-1 CD8+ T Cells

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To generate antigen-specific CD8⁺ T cells for adoptive cell transfer, splenocytes from Pmel-1 (Thy1.1⁺) TCR transgenic mice were depleted of erythrocytes by ACK lysis and cultured in complete media with 30 IU/ml recombinant human Interleukin-2 (rhIL2; Novartis) in the presence of 1 μM hgp100_25–33 peptide and expanded for 5 days.

Acquavella N., Clever D., Yu Z., Roelke-Parker M., Palmer D.C., Xi L., Pflicke H., Ji Y., Gros A., Hanada K.I., Goldlust I.S., Mehta G.U., Klebanoff C.A., Crompton J.G., Sukumar M., Morrow J.J., Franco Z., Gattinoni L., Liu H., Wang E., Marincola F., Stroncek D.F., Lee C.C., Raffeld M., Bosenberg M.W., Roychoudhuri R, & Restifo N.P. (2014). Type-1-cytokines synergize with oncogene inhibition to induce tumor growth arrest. Cancer immunology research, 3(1), 37-47.

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Expansion of CD3-depleted PBMCs

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CD3-depleted peripheral mononuclear cells (PBMCs) were isolated using a RosetteSepTM Human CD3 Depletion Cocktail (STEMCELL Technologies, Vancouver, BC, Canada). CD3-depleted PBMCs were placed in a T25 culture flask (Corning, Steuben, NY, USA) containing AIM-V medium (Life Technologies, New York, NY, USA) supplemented with 5% autologous plasma, 50 ng/mL recombinant human (rh) interleukin (rhIL)-18 (Medical and Biological Laboratories Co., Ltd.; MBL, Nagoya, Japan), and 3000 IU/mL rhIL-2 (Novartis, Basel, Switzerland) at 37 °C in a humidified 5% CO₂-containing atmosphere for 14 days. AIM-V supplemented with 3000 IU/mL IL-2 was replenished as necessary.

Shida Y., Nakazawa T., Matsuda R., Morimoto T., Nishimura F., Nakamura M., Maeoka R., Yamada S., Nakagawa I., Park Y.S., Yasukawa M., Tojo T., Tsujimura T, & Nakase H. (2021). Ex Vivo Expanded and Activated Natural Killer Cells Prolong the Overall Survival of Mice with Glioblastoma-like Cell-Derived Tumors. International Journal of Molecular Sciences, 22(18), 9975.

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Th17 Cell Differentiation Assay

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CD4⁺ T cells from WT, lmp7^−/− or lmp2^−/− mice were isolated from spleen and lymph nodes by negative magnetic cell sorting (MACS, Miltenyi Biotec). CD4⁺ T cells were cultured containing plate-bound 5 μg/ml anti-CD3 (clone 145-2C11) and 1 μg/ml soluble anti-CD28 (clone 37.51). For Th17 differentiation, cultures were supplemented with 5 μg/ml α-IFN-γ (clone XMG1.2), α-IL-4 (10% culture supernatants of clone 11B11), 50 U/ml rhIL-2 (Novartis), 1ng/ml rhTGF-β1 (PeproTech) and 40 ng/ml IL-6 (PeproTech). 200-400 nM ONX-0914 (Onyx Pharmaceuticals) or 300-400 nM BAY 11-7082 (Sigma-Aldrich) were added to the Th17 differentiation medium for indicated time points. Cells were restimulated with 750 ng/ml of ionomycin, 50 ng/ml of PMA in presence of 10 µg/ml Brefeldin A (all three substances, Sigma-Aldrich) and were analysed for IL-17A (clone eBio17B7, eBioscience) and IFN-γ (clone XMG1-2, eBioscience) production by intracellular staining (ICS). In some experiments, expression of transcription factor IRF4 (clone 3E4, eBioscience) was analysed by ICS.

Vachharajani N., Joeris T., Luu M., Hartmann S., Pautz S., Jenike E., Pantazis G., Prinz I., Hofer M.J., Steinhoff U, & Visekruna A. (2017). Prevention of colitis-associated cancer by selective targeting of immunoproteasome subunit LMP7. Oncotarget, 8(31), 50447-50459.

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Generation of Alloantigen-Specific T Cells

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Polyclonal CD4-transduced cells were obtained as previously described.²⁰ (link) CD4⁺ΔNGFR⁺ cells were beads-sorted using CD271⁺ Microbeads (Miltenyi Biotec) and expanded in X-VIVO15 medium with 5% human serum (BioWhittaker-Lonza), 100 U/mL penicillin-streptomycin (BioWhittaker), and 50 U/mL rhIL-2 (PROLEUKIN; Novartis). To generate alloantigen-specific transduced cells, we stimulated naive CD4⁺ T cells (10⁶/well) with allogeneic mDCs (10⁵/well). At days 7 and 10, medium was replaced by fresh medium supplemented with 25 U/mL rhIL-2. At day 14, cells were collected, washed, and 24 hr after the secondary stimulation with the same allo-mDCs used for priming, cells were transduced with LV-GFP/ΔNGFR (allo-CD4^ΔNGFR) or LV-IL-10/ΔNGFR (allo-CD4^IL-10) at an MOI of 20. Transduced CD4⁺ΔNGFR⁺ T cells were purified and stimulated every 2 weeks with allogeneic feeder mixture as previously described.²⁰ (link) Purity of selected cells was consistently greater than 95%. Allo-CD4^ΔNGFR and allo-CD4^IL-10 cells after two to three feeder mixture expansions were used to perform in vitro and in vivo experiments.

Locafaro G., Andolfi G., Russo F., Cesana L., Spinelli A., Camisa B., Ciceri F., Lombardo A., Bondanza A., Roncarolo M.G, & Gregori S. (2017). IL-10-Engineered Human CD4+ Tr1 Cells Eliminate Myeloid Leukemia in an HLA Class I-Dependent Mechanism. Molecular Therapy, 25(10), 2254-2269.

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PBMC Isolation and Pre-Stimulation

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Peripheral blood mononuclear cells (PBMC) were separated by density gradient from the peripheral blood of healthy donors after informed consent (Lymphoprep; Fresenius, Axis-Shield, Oslo, Norway) and then plated in RPMI 1640 with 1% FBS, 1% glutamine and 1% penicillin-streptomycin. Non-adherent cells were collected and pre-stimulated for 48 hours in RPMI 1640 supplemented with 10% heat-inactivated defined FBS, 500 UI/mL rhInterleukin-2 (rhIL-2, Proleukin, Novartis Farma S.p.a) and 7 μg/mL Phytohemagglutinin (PHA-M, Sigma-Aldrich) at the concentration of 1 × 10⁶ cells/mL.

Prapa M., Caldrer S., Spano C., Bestagno M., Golinelli G., Grisendi G., Petrachi T., Conte P., Horwitz E.M., Campana D., Paolucci P, & Dominici M. (2015). A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing. Oncotarget, 6(28), 24884-24894.

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Isolation and Cultivation of Murine and Human Immune Cells

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NK cells were isolated from splenocytes using the MACS NK cell separation kit (anti-DX5 microbeads, Miltenyi Biotec). Murine primary NK cells, YAC-1, RMA-S, RMA-Rae1, Stat1^−/− v-abl⁺ leukemic and B16F10 melanoma cells were cultivated as previously described.¹ (link) Human Jurkat cells were maintained in RPMI-1640 medium containing L-glutamine (PAA), 10% fetal calf serum (FCS, PAA), 50 µM 2-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin and 100 µg/mL streptomycin (Life Technologies). Lymphokine-activated killer (LAK) cells were maintained in 5,000 U/mL rhIL-2 (Proleukin, Novartis) for 5–7 d and treated with 250 U/mL rmIFN-β (PBL) for 4 h prior to gene analysis. Stat1^ind LAK cells were continuously cultivated in the presence of 50 ng/mL doxycycline.

Putz E.M., Majoros A., Gotthardt D., Prchal-Murphy M., Zebedin-Brandl E.M., Fux D.A., Schlattl A., Schreiber R.D., Carotta S., Müller M., Gerner C., Decker T, & Sexl V. (2016). Novel non-canonical role of STAT1 in Natural Killer cell cytotoxicity. Oncoimmunology, 5(9), e1186314.

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