Recombinant human il 2

Manufactured by R&D Systems

Sourced in United States, United Kingdom, Canada, China

Recombinant human IL-2 is a cytokine produced in E. coli cells. It has a molecular weight of approximately 15.5 kDa.

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Recombinant IL-2 (44 citations) Human IL-2 (33 citations) Recombinant human IL-2 protein (8 citations) Recombinant human interleukin 2 (IL-2) (3 citations) Human recombinant interleukin (IL)-2 (2 citations)

122 protocols using recombinant human il 2

Lentiviral Knockdown of NOX2 in CD8+ T Cells

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Healthy CD8⁺ T cells were infected separately with an empty shRNA vector control (sh-C, pLKO.1-puro) or four different human NOX2 shRNA (sh-1: NM-000397.2-93s1c1, TRCN0000064590; sh-2: NM-000397.2-399s1c1, TRCN0000064588; sh-3: NM-000397.2-813s1c1, TRCN0000064591; sh-4: NM-000397.2-1637s1c1, TRCN0000064589) lentiviral transduction particles (Sigma-Aldrich, St Louis, MO, USA), according to the manufacturer's instructions. Recombinant lentiviral particles were produced by transient transfection of 293FT cells as described60 (link). CD8⁺ T cells were infected with lentiviral particles22 (link)61 (link). Briefly, after stimulation with anti-CD3/28 antibodies (both 2 μg ml⁻¹) and human recombinant IL-2 (R&D Systems, Minneapolis, MN, USA; 1 ng ml⁻¹) for 24 h, CD8⁺ T cells were infected by lentiviral particles by centrifugation at 2,300 r.p.m for 60 min at room temperature in the presence of polybrene (8 μg ml⁻¹). These cells were replaced with fresh media containing human recombinant IL-2 (R&D Systems, Minneapolis, MN, USA; 1 ng ml⁻¹) for additional 48 h, and then selected with puromycin (1 μg ml⁻¹) for 7–10 days. The survival cells were selected using dead cell removal kit (Miltenyi Biotec, San Diego, CA, USA). The selected cells were tested to confirm diminished NOX2 expression by western blot analysis.

Bai A., Moss A., Rothweiler S., Serena Longhi M., Wu Y., Junger W.G, & Robson S.C. (2015). NADH oxidase-dependent CD39 expression by CD8+ T cells modulates interferon gamma responses via generation of adenosine. Nature Communications, 6, 8819.

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Lentiviral Knockdown of ASM in CD4+ T Cells

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Healthy CD4⁺ T cells were infected separately with an empty shRNA vector control (sh-C, pLKO.1-puro) or different human ASM shRNA (sh-1: NM_000543.2-367s1c1, TRCN0000049014; sh-2: NM_000543.2-1467s1c1, TRCN0000049015) lentiviral transduction particles (Invitrogen), according to the manufacturer's instructions. Recombinant lentiviral particles were produced by transient transfection of 293FT cells as described.^{40 (link)} CD4⁺ T cells were infected with lentiviral particles according to previously described method with a minor modification.^{41 (link)} Briefly, after stimulation with anti-CD3/28 antibodies (both 2 μg/ml) and human recombinant IL-2 (R&D Systems) (1 ng/ml) for 24 h, CD4⁺ T cells were infected by lentiviral particles by centrifugation at 2300 r.p.m. for 60 min at room temperature in the presence of polybrene (8 μg/ml). These cells were replaced with fresh media containing human recombinant IL-2 (R&D Systems) (1 ng/ml) for additional 48 h, and then selected with puromycin (2.25 μg/ml) for 5–10 days. The surviving cells were selected using a Dead Cell Removal Kit (Miltenyi Biotec, San Diego, CA, USA).

Bai A., Kokkotou E., Zheng Y, & Robson S.C. (2015). Role of acid sphingomyelinase bioactivity in human CD4+ T-cell activation and immune responses. Cell Death & Disease, 6(7), e1828-.

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Lentiviral Transduction of CD4+ T Cells

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CD4⁺ T cells purified from healthy human blood were infected separately with lentiviral transduction particles (Life Technologies) containing an empty shRNA vector control (sh-C, pLKO.1-puro), or four different human ASM shRNAs (sh-1: NM_000543.2-367s1c1, TRCN0000049014; sh-2: NM_000543.2-1054s1c1, TRCN0000049016; sh-3: NM_000543.2-1467s1c1, TRCN0000049015; sh-4: NM_000543.2-1225s1c1, TRCN0000049017), according to the manufacturer’s instructions. Recombinant lentiviral particles were produced by transient transfection of 293FT cells as described (28 (link)). CD4⁺ T cells were infected with lentiviral particles according to previously described method with minor modification (29 (link)). Briefly, after stimulation with anti-CD3/CD28 antibodies (both at 2 µg/ml) and human recombinant IL-2 (R&D Systems) (1 ng/ml) for 24 hr, CD4⁺ T cells were infected by lentiviral particles by centrifugation at 2300 rpm for 1hr at room temperature in the presence of polybrene (8 µg/ml). These cells were replenished with fresh media containing human recombinant IL-2 (R&D Systems) (1 ng/ml) for 48 hr, followed by selection with puromycin (2.25 µg/ml) for additional 5 to 10 days. The surviving cells were collected using the Dead Cell Removal Kit (Miltenyi Biotec, San Diego, CA) to deplete apoptotic cells, followed by examination of ASM protein expression using Western blot analysis.

Bai A., Moss A., Kokkotou E., Usheva A., Sun X., Cheifetz A., Zheng Y., Longhi M.S., Gao W., Wu Y, & Robson S.C. (2014). CD39 and CD161 modulate T helper type 17 responses in Crohn's disease. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3366-3377.

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PBMCs Isolation and T Cell Activation

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Venous blood was collected in EDTA tubes and PBMCs were isolated using a Ficoll-Hypaque gradient according to standard protocol (Axis-Shield Diagnostics, Dundee, UK). PBMCs were labeled with CellTrace Violet (ThermoFisher Scientific, Bleiswijk, the Netherlands) and were stimulated with phytohemagglutinin (PHA, 5 µg/ml, ThermoFisher Scientific), ConA (10 µg/ml), anti-CD3 (0.5 µg/ml, Sanquin, Amsterdam, the Netherlands) or anti-CD3/CD28 stimulator beads (0.5 bead per PBMC) with or without recombinant human IL-2 (1, 10 or 100 IU/ml, R&D Systems, Minneapolis, MN, USA) or IL-15 (1, 10 or 100 µg/ml, R&D Systems) for the indicated time-points. In some experiments, tofacitinib (Pfizer, New York, NY, USA; 200 or 1000 µM) was added to the anti-CD3/CD28 stimulated conditions. Cells were cultured in Iscove’s modified Dulbecco’s medium (ThermoFisher Scientific) supplemented with heat-inactivated fetal calf serum, Glutamax (ThermoFisher Scientific), 2-mercaptoethanol, penicillin, and streptomycin. For retroviral viral transduction, PBMCs from a healthy donor were isolated and T cell blasts were generated by culturing PBMCs with 1 µg/ml PHA (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 72 h in RPMI-1640 medium supplemented with 10% human serum (#H4522, Sigma-Aldrich) and expanded in culture with 100 IU/ml recombinant human IL-2 (R&D Systems).

Joosse M.E., Charbit-Henrion F., Boisgard R., Raatgeep R.(., Lindenbergh-Kortleve D.J., Costes L.M., Nugteren S., Guegan N., Parlato M., Veenbergen S., Malan V., Nowak J.K., Hollink I.H., Mearin M.L., Escher J.C., Cerf-Bensussan N, & Samsom J.N. (2021). Duplication of the IL2RA locus causes excessive IL-2 signaling and may predispose to very early onset colitis. Mucosal Immunology, 14(5), 1172-1182.

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Human T Cell Activation and Cytokine Profiling

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To activate the human T cells, the cells were thawed and cultured at 1 × 10⁶ cells/mL with a 2:1 ratio of αCD3/CD28 beads (Thermo Fisher) to T cells for 72 h in T cell media supplemented with 100 IU/mL recombinant human IL2 (R&D Systems). The T cells were then separated from the beads using a magnet, washed, and then resuspended at 1 × 10⁶ cells/mL for future experiments.
For in vitro cytokine experiments, human T cells were cultured at 1 × 10⁶ cells/mL for 72 h in T cell media alone or supplemented with 100 IU/mL recombinant human IL2, 15 ng/mL recombinant human IL7 (R&D Systems), or 20 ng/mL I recombinant human IL15 (R&D Systems). The following cytokines (all from R&D systems) were studied at the following concentrations (based on the literature): IL18 (20 and 100 ng/mL), IL6 (10 ng/mL), IL9 (10 ng/mL), IL12 (15 ng/mL), IL21 (15 ng/mL), IL27 (15 ng/mL), IL33 (10 ng/mL), and TNFα (10 ng/mL). At 48 h, the T cells cultured in the various cytokines were collected, counted, and resuspended at 1 × 10⁶ cells/mL in fresh media with fresh cytokines.

Tilsed C.M., Sadiq B.A., Papp T.E., Areesawangkit P., Kimura K., Noguera-Ortega E., Scholler J., Cerda N., Aghajanian H., Bot A., Mui B., Tam Y., Weissman D., June C.H., Albelda S.M, & Parhiz H. (2024). IL7 increases targeted lipid nanoparticle–mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation. Proceedings of the National Academy of Sciences of the United States of America, 121(13), e2319856121.

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Expansion of NK Cells using K562 Feeder Cells

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NK cells were expanded for 14 days using K562-mbIL21-41BBL feeder cells as described previously [58 (link)]. On day 0, 5 million mononuclear cells, separated from a buffy coat with Ficoll-Paque gradient centrifugation, were suspended in 40 ml RPMI-1640 with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L- glutamine, and 100 U/mL penicillin with 100 mg/mL streptomycin (PS) (referred to as R10), supplemented with 10 ng/ml recombinant human IL-2 (R&D Systems, 202-IL-050) together with 10 million irradiated (100 Gy) K562-mbIL21-41BBL feeder cells. After 7 days of culture and two passages, additional feeder cells were added in a 1:1 ratio. After 14 days of culture, NK cells were purified using an NK Cell Isolation Kit (Miltenyi) and frozen. Before the scRNAseq co-culture experiments, NK cells were thawed and cultured in R10 supplemented with 10 ng/ml recombinant human IL-2 (R&D Systems, 202-IL-050) 3 days before the experiments.

Huuhtanen J., Adnan-Awad S., Theodoropoulos J., Forstén S., Warfvinge R., Dufva O., Bouhlal J., Dhapola P., Duàn H., Laajala E., Kasanen T., Klievink J., Ilander M., Jaatinen T., Olsson-Strömberg U., Hjorth-Hansen H., Burchert A., Karlsson G., Kreutzman A., Lähdesmäki H, & Mustjoki S. (2023). Single-cell analysis of immune recognition in chronic myeloid leukemia patients following tyrosine kinase inhibitor discontinuation. Leukemia, 38(1), 109-125.

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HIV Latency Reactivation in Cell Lines

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HEK293T and CEM T cells were maintained in DMEM and RPMI 1640, respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin and 2 mM L-glutamine (Invitrogen, San Diego, CA, United States). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Paque gradient centrifugation (Amersham Biosciences) and treated with 10 ng/ml PHA-P (Phytohemagglutinin-P, Sigma Aldrich, St. Louis, MO, United States) for 48 h. The activated PBMCs (PHA-blasts) were cultured in RPMI 1640 with 10% FBS, antibiotics and 20 U/mL of human recombinant IL-2 (R&D systems).
For latent rfl-HIV reactivation, PMA (phorbol 12-myristate 13-acetate, Sigma Aldrich) plus Ionomycin (Cayman Chemical, Ann Arbor, MI, United States), or SAHA (Vorinostat, Sigma Aldrich) were used at 20 ng/ml, 1 and 5 μM, respectively.
Cellular DNA and RNA were extracted by the NucleoSpin RNA kit with RNA/DNA buffer set (Machery-Nagel, Düren, Germany) according to the manufacturer’s protocol.

Kobayashi-Ishihara M., Terahara K., Martinez J.P., Yamagishi M., Iwabuchi R., Brander C., Ato M., Watanabe T., Meyerhans A, & Tsunetsugu-Yokota Y. (2018). HIV LTR-Driven Antisense RNA by Itself Has Regulatory Function and May Curtail Virus Reactivation From Latency. Frontiers in Microbiology, 9, 1066.

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Generation and Activation of Epitope-Specific CTL

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E183-91 epitope-specific human CTL, C18-91, or E183-91 epitope-specific TCR-transduced human T cells were made as described^{40 (link),41 (link)}. The E183 CTL have not been authenticated and not been tested for mycoplasma. PBMC were freshly isolated by Ficoll-Paque PLUS (GE Healthcare Life Sciences) according to the manufacturer’s protocol and irradiated at 30 Gy. PBMC were resuspended in Aim-V medium with 2% human serum (Sigma-Aldrich) at a concentration of 2 × 10⁶ per ml. Human CTL were resuspended in the same medium at 10⁶ per ml. Totally, 500 μl of PBMC and 500 μl of human CTL were pooled per well in a 24-well plate. Final concentrations of 20 U ml⁻¹ human recombinant IL-2, 10 ng ml⁻¹ of IL-7, 10 ng ml⁻¹ of IL-15 (R&D Systems) were added to the human CTL culture. Lectin from Phaseolus vulgaris (Sigma-Aldrich) was added to the human CTL culture at a concentration of 1.5 μg ml⁻¹. PBMC were collected from healthy volunteers under the protocol approved by NUS IRB. Informed consent was obtained from all donors. GAG-A2-specific TCR-transduced T cells were made as described^{27 (link)}.

Zhao X., Sankaran S., Yap J., Too C.T., Ho Z.Z., Dolton G., Legut M., Ren E.C., Sewell A.K., Bertoletti A., MacAry P.A., Brzostek J, & Gascoigne N.R. (2018). Nonstimulatory peptide–MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling. Nature Communications, 9, 2716.

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Allogenic T Cell Polarization by DCs

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The capacity of treated and non-treated DCs to induce T cell polarization was assessed in an allogenic stimulation assay. Naïve allogenic T cells (1×10⁵/well) were co-cultivated with DCs (0.5x10⁴/well) in 96-well round-bottom plate in a final volume of 200 μl, for 6 days. For the flow cytometric detection of intracellular cytokines, the co-cultures were treated with phorbol myristate acetate (PMA) (20 ng/ml), inomycin (500 ng/ml) and monensin (3 µM) (all from Sigma-Aldrich) for the last 3 h of incubation. Cells were analyzed for the production of cytokines (IL-4, IL-10 and IFN-γ) by flow cytometry analysis (BD FACS LSR II flow cytometer).
To analyze the capacity of stimulated DCs to induce regulatory T cells (Tregs), allogenic naïve T cells were primed with DCs at a 1:50 DC/T cell ratio for 5 days. Allogenic T cells (1×10⁵/well) were plated with DCs (0.2x10⁴/well) in 96-well round-bottom plate, and after 3 days of incubation, 20 IU⁄mL of human recombinant IL-2 (2 ng/ml, R&D Systems) were added. The culture was incubated the next 48 hours and for the last 4 h of incubation, the co-cultures were treated with PMA/ionomycin and monensin. The expression of CD4, CD25 and FoxP3 and the percentage of CD4⁺ CD25⁺ T cells producing cytokines IL-10 and TGF-β was determined by flow cytometry.

Ilic N., Bojic-Trbojevic Z., Lundström-Stadelmann B., Cujic D., Mitic I, & Gruden-Movsesijan A. (2022). Immunomodulatory components of Trichinella spiralis excretory-secretory products with lactose-binding specificity. EXCLI Journal, 21, 793-813.

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Isolation and Activation of Human CD4+ T Cells

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Human peripheral blood leukopaks (total n=46 different donors) were purchased from either HemaCare (Northridge, CA) (n=15 different donors) or iXSells Biotechnologies (San Diego CA) (n=31 different donors) who collected the leukopaks under either a Hemacare or iXSells Biotechnologies IRB-approved donor consent. Aliquots of the leukopaks were frozen in liquid nitrogen and thawed upon use for CD4 cell isolation. Human primary CD4+ cells were isolated and purified from the leukopak aliquots using a human CD4 T cell negative selection kit (StemCell Technologies). CD4+ cells were maintained in ImmunoCult human T cell expansion medium (StemCell Technologies) with 100 unit/ml human recombinant IL-2 (R&D Systems). To activate CD4+ T cells, 1:40 ImmunoCult human CD3/CD28 T cell activator (StemCell Technologies) was added to the medium, and expanded cells were diluted to 1 million cells per ml at day 3 and day 5 after activation by adding more ImmunoCult human T cell expansion medium with IL-2 (complete medium). CD4+ T cells were cultured for 3–5 days before transfecting with the CBE single base editor mRNA and gRNA.

Weng N., Miller M., Pham A.K., Komor A.C, & Broide D.H. (2021). Single base editing of rs12603332 on Chromosome 17q21 with a Cytosine Base Editor regulates ORMDL3 and ATF6α expression. Allergy, 77(4), 1139-1149.

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