Ril 2

Manufactured by R&D Systems

Sourced in United States

The RIL-2 is a laboratory instrument designed for the rapid isolation and purification of recombinant proteins. It utilizes a proprietary affinity-based chromatography system to capture and purify target proteins from complex biological samples.

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Lab products found in correlation

Ril 7, by R&D Systems (7 mentions) Hepes, by Merck Group (5 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions) Ril 33, by R&D Systems (4 mentions) Dynabead, by Thermo Fisher Scientific (4 mentions) Tgf β, by R&D Systems (3 mentions) L glutamine, by Thermo Fisher Scientific (3 mentions) Rpmi 1640 medium, by Merck Group (3 mentions) Sodium pyruvate, by Thermo Fisher Scientific (3 mentions) Golgiplug, by BD (3 mentions) Anti cd3, by Thermo Fisher Scientific (2 mentions) Soluble anti cd28, by Thermo Fisher Scientific (2 mentions) L glutamine, by Merck Group (2 mentions) Ficoll paque plus, by GE Healthcare (2 mentions) Human ab serum, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thomas Scientific (2 mentions) Antimycin a, by Merck Group (2 mentions) Rmil 33, by R&D Systems (2 mentions) Rotenone, by Merck Group (2 mentions) Extracellular flux analyzer, by Agilent Technologies (2 mentions)

Spelling variants of this product

Human rIL-2 (2 citations)

37 protocols using ril 2

Murine and Human Naive CD4+ T Cell Differentiation

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Naïve CD4⁺ T cells were enriched from spleens of WT, SMYD3^−/− or Foxp3^eGFP mice as described ^{18 (link)}. Cells stimulated for 48 hours to 5 days using plate-bound anti-CD3 (2.5 μg/ml; eBioscience), soluble anti-CD28 (3 μg/ml; eBioscience) plus TGFβ (2 to 20 ng/ml) and rIL-2 (10 ng/ml; all from R&D Systems). Human naïve CD4⁺T cells were isolated as above and converted into iTreg cells using 5 ng/ml of TGFβ. For T helper (H) cell differentiation, naïve CD4⁺T cells were skewed towards TH17 using IL-6 (1 ng/ml) and TGFβ (2 ng/ml) plus anti-IFNγ (10 μg/ml), anti-IL12 (10 μg/ml), anti-IL4 (10 μg/ml), and anti-CD28 (3 μg/ml).

de Almeida Nagata D.E., Ting H.A., Cavassani K.A., Schaller M.A., Mukherjee S., Ptaschinski C., Kunkel S.L, & Lukacs N.W. (2015). Epigenetic control of Foxp3 by SMYD3 H3K4 histone methyltransferase controls iTreg development and regulates pathogenic T cell responses during pulmonary viral infection. Mucosal immunology, 8(5), 1131-1143.

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Isolation and Culture of PBMCs

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With approval from the IRB of the National University of Singapore, peripheral blood was obtained from healthy donors following written informed consent and all experiments were performed in accordance to relevant guidelines and regulations. HLA of donors were identified using sequence based typing as described previously^{43 (link)}. PBMCs were isolated with Ficoll-Paque PLUS (GE Healthcare) and maintained in RPMI 1640 supplemented with 5% human AB serum (Sigma), 100 IU/ml penicillin-streptomycin (Gibco), 2 mM L-glutamine (Sigma) and 10 mM HEPES (Sigma). For 14-day cultures, PBMCs (1 × 10⁶ cells/ml) were incubated with 10 µM peptides (GenScript) and supplemented with 25 U/ml rIL2 (R&D Systems). Culture medium with rIL2 was replenished every 2–3 days from Day 5 onwards.

Xiao Z., Ye Z., Tadwal V.S., Shen M, & Ren E.C. (2017). Dual non-contiguous peptide occupancy of HLA class I evoke antiviral human CD8 T cell response and form neo-epitopes with self-antigens. Scientific Reports, 7, 5072.

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Profiling IL-33-Activated Innate Lymphoid and T Cells

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Foxp3^eGFP reporter mice were treated with 100 ng of rm IL-33 (R&D Systems) i.n. every 3 days for 2–3 weeks. Lin⁻ CD45⁺CD90⁺CD25⁺IL-33R⁺ ILC2s and/or CD45⁺CD3⁺CD5⁺CD4⁺Foxp3⁻IL-33R⁺ T_H2 cells were sort-purified from the lung and rested for 18 h in DMEM Complete Media (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Denville Scientific), 1% l-glutamine (GIBCO), 1% penicillin/streptomycin (GIBCO), 25 mm HEPES buffer, and 55 μm 2-β-mercaptoethanol (Sigma-Aldrich)) on ice. For Arg1 inhibitor studies only, cells were then cultured for additional 24 hin DMEM Complete Media with 20 ng/ml rIL-2, 20 ng/ml rIL-7 and 50 ng/ml rIL-33 (all cytokines from R&D Systems) at 37°C. Cells were plated at 200,000 cells per well and OCR and ECAR measured in XF media (non-buffered RPMI 1640 containing 25 mM glucose, 2 mM l-glutamine, and 1 mM sodium pyruvate) under basal conditions, in response to 1 μM oligomycin, 1.5 μM fluoro-carbonyl cyanicide phenylhydrazone (FCCP) and 100 nM rotenone + 1 μM antimycin A (Sigma-Aldrich) and as indicated after DMSO or 500 μM nor-NOHA injection using a 96 well Extracellular Flux Analyzer (Seahorse Bioscience).

Monticelli L.A., Buck M.D., Flamar A.L., Saenz S.A., Wojno E.D., Yudanin N.A., Osborne L.C., Hepworth M.R., Tran S.V., Rodewald H.R., Shah H., Cross J.R., Diamond J.M., Cantu E., Christie J.D., Pearce E.L, & Artis D. (2016). Arginase 1 is an innate lymphoid cell-intrinsic metabolic checkpoint controlling type 2 inflammation. Nature immunology, 17(6), 656-665.

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Electroporation of Activated PBMCs

Cited 1 time

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PBMCs were collected under informed consent from healthy donors. PBMCs were stimulated with 600 U/ml IL-2 (rIL-2; R&D Systems, Minneapolis, MN) and 50 ng/ml anti-CD3 (OKT-3; eBioscience, San Diego, CA) in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum for 7 days. The concentration of rIL-2 was increased to 1,000 IU/ml on day 8.
Electroporation were performed with the Nucleofector device II (Lonza, Cologne, Germany). 10 × 10⁶ PBMCs were activated for 8 days as described above and were thereafter re-suspended in 100 μL of Cell Line Nucleofector Solution V (Lonza, Cologne, Germany) and TCR mRNA was added at 200 μg/ml47 (link). The mixture was placed in a certified cuvette (Lonza, Cologne, Germany) and electroporated. After electroporation, cells were re-suspended in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum and 100 IU/ml rIL-2. Transfected cells were maintained in a humidified 37 °C and 5% CO₂ incubator until flow cytometry analysis and/or co-culture experiments.

Holmström F., Chen M., Balasiddaiah A., Sällberg M., Ahlén G, & Frelin L. (2016). Functional differences in hepatitis C virus nonstructural (NS) 3/4A- and 5A-specific T cell responses. Scientific Reports, 6, 24991.

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Naive CD4+ T Cell Isolation and Activation

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Naïve CD4⁺ T cells were isolated from B10.PLxC57BL/6 mice by MACS according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany and cells were cultured at a density of 3 × 10⁶/ml on six-well plates for five days in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 × 10^-5 M 2-ME (all from Invitrogen Life Technologies, Paisley, UK), and 10% FCS (Sigma, Irvine, Scotland For T-cell stimulation, cells were cultured at a density of 1.5 × 10⁶/ml in the presence or absence of 10 U/ml rIL-2 (R&D Systems, Abingdon, Oxfordshire, UK on six-well plates pre-coated with 2 μg/ml anti-CD3 (clone 145.2C11; eBioscience, Hatfield, Hertfordshire, UK) plus anti-CD28 (clone 37.51; e-Bioscience).

Nestor C.E., Ottaviano R., Reinhardt D., Cruickshanks H.A., Mjoseng H.K., McPherson R.C., Lentini A., Thomson J.P., Dunican D.S., Pennings S., Anderton S.M., Benson M, & Meehan R.R. (2015). Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems. Genome Biology, 16(1), 11.

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Expansion of Cytotoxic T-Cells In Vitro

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Isolated CD8+ T-cells were suspended at a concentration of 10⁶ cells/mL in culture medium (RPMI 1640-containing L-glutamine, 1% penicillin/streptomycin/amphotericin B, 1% HEPES, 1% non-essential amino acids, 1% sodium pyruvate, 0.05mM 2-mercaptoethanol, and 10% fetal bovine serum). Cells were mixed with Dynabeads (Gibco, ThermoFisher) at a bead-to-cell ratio of 1:1 and 100 U/ml rIL-2 (R&D Systems). Cells were incubated at 37°C with 5% CO₂ in a standard incubator. Cells were examined daily, and after day 3, cells were split daily. Beads were removed on day 3 (for human cells) or day 5 (for murine cells), and then maintain in media with 100 U/ml rIL-2.

Vesper S.J., Magnuson M.L., Dearborn D.G., Yike I, & Haugland R.A. (2001). Initial Characterization of the Hemolysin Stachylysin from Stachybotrys chartarum. Infection and Immunity, 69(2), 912-916.

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Activation of CD4+ T Cells

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CD4⁺ cells were cultured in 96-well round-bottom plates (Corning, Corning, NY) at 50,000 cells per well in advanced RPMI 1640 (Life Technologies/Fisher Scientific, Waltham, MA) supplemented with 5% Australian-produced heat-inactivated FBS, 55 µM 2-ME, 100 U/ml and 100 µg/ml penicillin/streptomycin, and 0.2 mM l-glutamine (Life Technologies/Fisher Scientific, Waltham, MA) at 5% CO₂, 37°C. For CD4⁺ activating conditions, wells were coated with anti-CD3ε (eBioscience, San Diego, CA) at 2.5 µg/ml in PBS, incubated at 4°C overnight, and washed twice with PBS before receiving cells. Where indicated, cultures were supplemented with rIL-2 (R&D Systems, Minneapolis, MN) with and without IL-4 (R&D Systems, Minneapolis, MN), with the equimolar final concentration being 728 pM.

Zhou J.Y., Glendenning L.M., Cavanaugh J.M., McNeer S.K., Goodman W.A, & Cobb B.A. (2023). Intestinal Tr1 Cells Confer Protection against Colitis in the Absence of Foxp3+ Regulatory T Cell–Derived IL-10. ImmunoHorizons, 7(6), 456-466.

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Cytotoxic Effects of NK Cells on HNSCC

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We used two HNSCC cell lines in this study. Cal27 cell line (tongue origin) was purchased from ATCC and was maintained in Dulbecco’s Modified Eagle Medium (Sigma) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma). JHU-29 (tongue) cell line was procured from the Johns Hopkins University, and was maintained in RPMI-1640 medium (Sigma) supplemented with 10% FBS and 1% penicillin/streptomycin. The human NK cell line (NK3.3) was cultured in RPMI-1640 medium supplemented with 10% FBS, 1% glutamine, 1% penicillin-streptomycin, and 200 IU/ml recombinant IL-2 (rIL-2) (R & D Systems) (16 (link)). We added IL-12 overnight to the NK 3.3 cells, then removed residual IL-2 by washing, exposed with BME (1% v/v) for additional 20 h before incubating with cancer cells. HNSCC cells were co-cultured with BME treated NK3.3 cells at different Tumor Cell/Target: Effector Cell (T:E) (1:10) ratios for 24 hr. Cytotoxicity was measured by using a multiTox-fluor multiplex cytotoxicity assay kit (Promega) following the manufacturer’s protocol, and readings were taken using a Bio-Tek plate reader.

Bhattacharya S., Muhammad N., Steele R., Kornbluth J, & Ray R.B. (2017). Bitter Melon Enhances Natural Killer Mediated Toxicity against Head and Neck Cancer Cells. Cancer prevention research (Philadelphia, Pa.), 10(6), 337-344.

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Hamster-Derived Anti-PD-1 Antibody Protocol

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MH5A was previously developed by immunizing Armenian hamsters with a mouse PD-1HIg fusion protein and Freund’s adjuvant as previously described in detail (22 (link)). Control hamster IgG was purchased from Rockland Immunochemicals (Gilbertsville, PA). Fluorescently labeled antibodies for flow cytometry including CD4, CD8, H-2K^d, IFN-γ, CD19, CD25, Gr-1, CD11b, DX5, and anti-hamster IgG-biotin were purchased from BD Biosciences (San Jose, CA). CD3 and CD28 mAbs for cell culture were also purchased from BD Biosciences. AnnexinV and 7-AAD were purchased from BD Biosciences. FoxP3-APC, FoxP3-FITC, and streptavidin-APC and PerCP, and TNF-α ELISA were purchased from eBioscience (San Diego, CA). Ki67-APC and ZombieNIR were purchased from Biolegend. CFSE (Vybrant CFDA cell tracer kit) was purchased from Life Technologies. TGF-β and rIL-2 were purchased from R&D Systems (Minneapolis, MN).

Flies D.B., Higuchi T, & Chen L. (2015). Mechanistic Assessment of PD-1H Coinhibitory Receptor-Induced T-Cell Tolerance to Allogeneic Antigens. Journal of immunology (Baltimore, Md. : 1950), 194(11), 5294-5304.

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Culturing Lung and Skin ILC2s

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Lin⁻CD45⁺Thy1⁺Red5⁺ ILC2s from lung and skin were FACS sorted and 2500 (lung) or 1000 (skin) cells per well were cultured in RPMI-1640 (Sigma) containing 10% FBS, 10 mM HEPES (Sigma), 100 μM non-essential amino acids (Sigma), 1 mM sodium pyruvate (Gibco), 100 μU/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 50 μM 2-mercaptoethanol (Gibco), 10 ng/ml rIL-7 (R&D Systems), 10 ng/ml rIL-2 (R&D Systems), and 10 ng/ml rIL-33 (BioLegend) in 96-well round bottom plates at 37 °C under 5% CO₂. After 3 days, supernatants were collected and the concentration of CXCL2 was measured by ELISA (ThermoFisher, EMCXCL2).

Schneider C., Lee J., Koga S., Ricardo-Gonzalez R.R., Nussbaum J.C., Smith L.K., Villeda S.A., Liang H.E, & Locksley R.M. (2019). Tissue-resident group 2 innate lymphoid cells differentiate by layered ontogeny and in situ perinatal priming. Immunity, 50(6), 1425-1438.e5.

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