Rhil 2

Manufactured by Miltenyi Biotec

Sourced in Germany

RhIL-2 is a recombinant human interleukin-2 product. Interleukin-2 is a cytokine that plays a crucial role in the immune system by promoting the growth and differentiation of T cells.

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Premium-grade rhIL-2 (2 citations)

29 protocols using rhil 2

Human PBMC Isolation and Immune Cell Culture

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Human peripheral blood mononuclear cells (PBMC) were isolated from the blood using Ficoll Paque Plus (Sigma-Aldrich). The subtypes of MHC I molecules of Human PBMC were examined and HLA-A2⁺ human PBMC were identified by flow cytometry. Primary T cells were activated and expanded by culturing 1 × 10⁶ PBMC in TexMACS medium (Miltenyi Biotec, Germany) supplemented with 500 IU/ml human recombinant Interleukin 2 (rhIL2) (Miltenyi Biotec), with 10 µl TransAct (Miltenyi Biotec) which contains anti-CD3 and CD28 antibodies, for 3 days. The T cells were then purified with a pan-T cell isolation kit (Miltenyi Biotec) and cultured in TexMACS medium + rhIL2 for 2 weeks before CD3/28 antibody re-stimulation or DC cell-based antigen stimulation. Primary NK cells were activated and expanded by culturing 1 × 10⁶ PBMC in NK MACS medium (Miltenyi Biotec) supplemented with 5% human AB serum (Invitrogen) and 500 IU/ml rhIL2, with 5 × 10⁵ microbeads conjugated with anti-CD335 and CD2 antibodies (Miltenyi Biotec) for 7 days. The NK cells were purified with NK cell isolation kit (Miltenyi Biotec) and cultured in NK MACS medium + human AB serum + rhIL2 for 1 week before hypoxic culture. The purity of the immune cells was examined by flow cytometry analysis.

Ma S., Zhao Y., Lee W.C., Ong L.T., Lee P.L., Jiang Z., Oguz G., Niu Z., Liu M., Goh J.Y., Wang W., Bustos M.A., Ehmsen S., Ramasamy A., Hoon D.S., Ditzel H.J., Tan E.Y., Chen Q, & Yu Q. (2022). Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy. Nature Communications, 13, 4118.

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Cytotoxic Capacity of NK Cells

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The cytotoxic capacity of NK cells was assessed using the E-Cadherin expressing breast cancer epithelial cell line MCF-10A as target. Briefly, 20,000 MCF-10A cells were plated/well of a 96 flat-bottom plate 24 hours prior to the cytotoxicity assay. On the day of the assay, MCF-10A cells were labeled with Calcein-AM (Sigma-Aldrich) at 10μM for 1 hour before co-culture with NK cells in complete medium containing 500 IU/ml rhIL-2 (Miltenyi Biotech) and as described elsewhere (34 (link)). NK cells transfected with siRNA specific for KLRG1 or scrambled control siRNA for 36 hours were pre-treated with the AMPK agonist A769662 (Tocris Bioscience) at 150 μM or equivalent DMSO as negative control for 2 hours before being added to the target cells. Effector and target cells were combined at a ratio of 40:1 in triplicate and cultured in complete medium containing 500 IU/ml rhIL-2 (Miltenyi Biotech) for 4 hours. After 4 hours of co-culture fluorescence was measured in 75 μl of cell culture supernatant using a Spectramax Gemini spectrofluorimeter. Specific lysis was calculated as % killing = (test release–spontaneous release) / (max release–spontaneous release) x 100.

Müller-Durovic B., Lanna A., Covre L.P., Mills R.S., Henson S.M, & Akbar A.N. (2016). Killer Cell Lectin-like Receptor G1 (KLRG1) inhibits NK cell function through activation of AMP-activated Protein Kinase. Journal of immunology (Baltimore, Md. : 1950), 197(7), 2891-2899.

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Expansion of Antigen-Specific T Cell Clones

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Retrovirally transduced T cells were produced and expanded as previously described (11 (link)). Briefly, after isolation, PBMCs were stimulated with 600 IU/mL of recombinant human interleukin-2 (rhIL-2; Miltenyi Biotec, 130-097-745) and 50 ng/mL of anti-CD3 (eBioscience, 16-0037-81) in AIM-V (ThermoFisher Scientific, 12055091), 2% (v/v) human AB serum (Sigma-Aldrich, H6914) for 48 h, before transduction with a MP-71 vector containing the Vα34 and Vβ3 chains of the HBs183-91-specific (Ts) or the HBcore18-27-specific (Tc) TCR sequences. Bulk transduced T cells were expanded and restimulated using 0.5–1 × 10⁶ TCR-transduced T cells, 2 × 10⁵ irradiated (2,500 rads) T2 cells pulsed with 1 µg/mL of either HBs183-91 peptide (FLLTRILTI) or HBcore18-27 (FLPSDFFPSV) (Genscript, custom synthesized peptides) and 1.8 × 10⁶ irradiated PBMCs as feeders. Cells were cultured for about 2 weeks in AIM-V, 2% human AB serum supplemented with 100 IU/mL of rhIL-2, 10 ng/mL of rhIL-7 (Miltenyi Biotec, 130-095-362), and 10 ng/mL of rhIL-15 (Miltenyi Biotec, 130-095-764). In some experiments, CD8⁺ T cells were enriched through negative selection of transduced T cells using CD4 microbeads (Miltenyi Biotec, 130-045-101) following the manufacturer’s instructions.

Lee S.W., Adriani G., Ceccarello E., Pavesi A., Tan A.T., Bertoletti A., Kamm R.D, & Wong S.C. (2018). Characterizing the Role of Monocytes in T Cell Cancer Immunotherapy Using a 3D Microfluidic Model. Frontiers in Immunology, 9, 416.

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Expansion of HIV/EBV-specific CD8+ T cells

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Thawed PBMCs from HIV-infected individuals were labeled with CellTrace Violet (Thermo Fisher Scientific) at 1μM according to the manufacturer's protocol. The cells were then plated at 5 × 10⁶ cells/well in 24 well plate in CellGenix GMP DC Medium (CellGenix) containing recombinant human (rh) IL-7 (1ng/ml, PeproTech) to support minimal T cell survival and rh FLT3L (50ng/ml, PeproTech) to mobilize primary DCs. After 24 hours (day 1), cognate CD8 epitope peptides from HIV or EBV were added to the culture at 1μg/ml. On day 2, human serum (Access Biologicals) and rh IL-2 (Miltenyi Biotec) were added at final concentrations of 8% (volume/volume) and 20U/ml, respectively. Half of the medium was replaced with RPMI-1640 containing 8% human serum, rhIL-2 (20U/ml), and penicillin-streptomycin (100 U/ml, Quality Biological) on days 5, 7 and 9. HIV and EBV-specific CD8⁺ T cells in the culture were analyzed by flow cytometry on days 6 and 12.

Takata H., Kakazu J.C., Mitchell J.L., Kroon E., Colby D.J., Sacdalan C., Bai H., Ehrenberg P.K., Geretz A., Buranapraditkun S., Pinyakorn S., Intasan J., Tipsuk S., Suttichom D., Prueksakaew P., Chalermchai T., Chomchey N., Phanuphak N., de Souza M., Michael N.L., Robb M.L., Haddad E.K., Crowell T.A., Vasan S., Valcour V.G., Douek D.C., Thomas R., Rolland M., Chomont N., Ananworanich J, & Trautmann L. (2022). Long-term antiretroviral therapy initiated in acute HIV infection prevents residual dysfunction of HIV-specific CD8+ T cells. eBioMedicine, 84, 104253.

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Electroporation-Mediated siRNA Knockdown in Freshly Isolated KLRG1+ NK Cells

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siRNA was introduced into freshly isolated KLRG1^bright NK cells by electroporation using the Amaxa® Human NK Cell Nucleofector® Kit and Nucleofector® technology (Lonza) according to the manufacturer’s instructions. Briefly, 3x10⁶ freshly isolated KLRG1^bright NK cells were resolved in Nucleofector® solution provided by the manufacturer and 200 nmol PP2C (PP2Cα siRNA, sc-42937, Santa Cruz Biotechnology) or 300 nmol KLRG1 siRNA (KLRG1 siRNA, sc-42937, Santa Cruz Biotechnology). Nucleofection was performed with the Nucleofector™ I Device Program U-01 (Lonza). Cells were then transferred into 12-well cell culture plates containing pre-equilibrated complete medium and incubated for 36 hours. NK cells transfected with KLRG1 siRNA were cultured in complete medium containing 500 IU/ml rhIL-2 (Miltenyi Biotech). Knock-down efficiency of KLRG1 siRNA was monitored by measuring cell surface expression of KLRG1 by flow cytometry (Fig. S3A). Knock-down of PP2Cα was confirmed by immunoblot analysis (rabbit-anti-PP2Cα mAb, clone D18C10, XP, from Cell Signaling Technology) (Fig. S3B). In all experiments a scrambled control siRNA (sc-37007, Santa Cruz Biotechnology) was used.

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In Vitro 3D Microfluidic Liver Co-Culture Model

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Collagen hydrogels with 2 × 10⁶ HepG2-PreS1-GFP or Hep3B-GFP cells/mL were prepared as previously described^{23 (link)} and injected into dedicated 3D regions of a commercially available microfluidic AIM Chip (AIM Biotech). The hydrogel was polymerized by thermal cross-linking at 37 °C for 30 min. Subsequently, media channels in the hydrogel were hydrated with R10 medium and incubated for 24 h to permit interaction between HepG2-PreS1-GFP and the hydrogel microenvironment. The medium in the microfluidic device was then replaced with AIM-V medium supplemented with 2% human AB serum, 100 IU/mL rhIL-2, and 3 μM DRAQ7 (Miltenyi Biotec) in preparation for the first confocal imaging. 9 × 10⁴ Electroporated HBV S183 antigen-redirected T cells or 6 × 10⁴ NK cells labeled with 3 μM CellTracker Violet BMQC (Invitrogen) were added to one channel of the device and incubated for 24 h before the next confocal image acquisition. For devices containing Hep3B-GFP cells, 9 × 10⁴ CD133 CAR-T cells were added per device without the need for labeling as they are also expressing mCherry.

Lam M.S., Reales-Calderon J.A., Ow J.R., Aw J.J., Tan D., Vijayakumar R., Ceccarello E., Tabaglio T., Lim Y.T., Chien W.L., Lai F., Tanoto A.T., Chen Q., Sobota R.M., Adriani G., Bertoletti A., Guccione E, & Pavesi A. (2023). G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma. Nature Communications, 14, 563.

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Engineered Cell Lines for Research

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HEK293T (ATCC) and MV4-11 (ATCC) were cultured in media per the supplier’s recommendation. Expi293 (Thermo Fisher Scientific) was cultured in media per the supplier’s recommendation. U87-EGFR is a kind gift from Cellectis SA (Paris, France). U87-EGFR was derived from the parental cell line, U87MG (ATCC) by first knocking out endogenous EGFR using Transcription Activator-Like Effector Nucleases (TALEN), and then stably overexpressing full-length human EGFR via lentiviral transduction. X-VIVO^TM 15 was obtained from Lonza, rhIL-2 from Miltenyi Biotech, human serum AB from Seralab, human T-activator CD3/CD28 from Thermo Fisher Scientific, MACS^® LD-column from Miltenyi Biotech, Hyclone FBS from GE Healthcare Life Sciences, Ficoll-Paque PLUS from GE Healthcare Life Sciences and Pan T cell Isolation Kit, human from Miltenyi Biotech. Methotrexate disodium salt and leucovorin calcium were obtained from Alfa Aesar and Sigma, respectively.

Park S., Pascua E., Lindquist K.C., Kimberlin C., Deng X., Mak Y.S., Melton Z., Johnson T.O., Lin R., Boldajipour B., Abraham R.T., Pons J., Sasu B.J., Van Blarcom T.J, & Chaparro-Riggers J. (2021). Direct control of CAR T cells through small molecule-regulated antibodies. Nature Communications, 12, 710.

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Expansion of SARS-CoV-2-specific T Cells

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SARS-CoV-2-specific T cells enriched via CCS using the CliniMACS Prodigy device and MACS GMP PepTivator SARS-CoV-2 Select (Miltenyi Biotec) were seeded into 24 well plates at 1 × 10⁵ cells per well in TexMACS (Miltenyi Biotec). For feeder cells, autologous PBMCs were irradiated at 50 Gy and seeded at 1 × 10⁷ cells per well. Cultures were supplemented with recombinant human Interleukin-2 (rhIL-2, 50 IU/ml; Miltenyi Biotec) and maintained for 7–13 days including regular media change and splitting of the cells.

Bonifacius A., Tischer-Zimmermann S., Santamorena M.M., Mausberg P., Schenk J., Koch S., Barnstorf-Brandes J., Gödecke N., Martens J., Goudeva L., Verboom M., Wittig J., Maecker-Kolhoff B., Baurmann H., Clark C., Brauns O., Simon M., Lang P., Cornely O.A., Hallek M., Blasczyk R., Seiferling D., Köhler P, & Eiz-Vesper B. (2022). Rapid Manufacturing of Highly Cytotoxic Clinical-Grade SARS-CoV-2-specific T Cell Products Covering SARS-CoV-2 and Its Variants for Adoptive T Cell Therapy. Frontiers in Bioengineering and Biotechnology, 10, 867042.

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Expansion of Autologous Vδ2 T Cells

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PBMCs from ALL patients and HV (healthy volunteers) were isolated by density gradient (Lymphoprep) and were then frozen until use. To expand autologous Vδ2 T cells, frozen PBMCs from ALL patients were stimulated with ZOL 1µM (Sigma-Aldrich, Saint Louis, MO) and rhIL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany) at day 0. From day (d) 5, rhIL-2 was renewed every 2 days and the cells were maintained at 1.5 × 10⁶/mL until d14. rh IL-15 (10 ng/mL; Miltenyi Biotec) was also added starting at d2 and was renewed every 2 days, as IL-15 has been shown to enhance the proliferative capacity of Vδ2 T cells [55 (link),56 (link),57 (link)]. Fresh autologous expanded Vδ2 T cells were then used for functional assays, depending on the quantity of Vδ2 T cells. The fold increase in viable Vδ2 T cells was calculated using the formula: (d14 %Vδ2 × d14 total cell number/(d0 % Vδ2 × d0 total cell number).

Le Floch A.C., Rouvière M.S., Salem N., Ben Amara A., Orlanducci F., Vey N., Gorvel L., Chretien A.S, & Olive D. (2023). Prognostic Immune Effector Signature in Adult Acute Lymphoblastic Leukemia Patients Is Dominated by γδ T Cells. Cells, 12(13), 1693.

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Isolation and Stimulation of Primary Immune Cells

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Peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll-Hypaque density gradient centrifugation. CD4⁺ and CD8⁺ T cells were purified by negative selection (Miltenyi Biotec) and stimulated with human CD3/CD28/CD2 antibody-coated beads (Miltenyi Biotec) at 1:1 bead-to-cell ratio, in the presence of 50 IU/ml of rhIL-2 (Miltenyi Biotec) for three days. CD19⁺ B cells were purified by positive selection (Miltenyi Biotec) and stimulated with 8 IU/ml of CD40L (Miltenyi Biotec) in the presence of 50 IU/ml of IL-4 (Miltenyi Biotec) for three days. CD14⁺ monocytes were purified by positive selection (Miltenyi Biotec) and stimulated with 100 ng/ml of LPS (eBioscience) and 500 ng/ml of R848 (InvivoGen) for three days. Primary cells were maintained in RPMI 1640 medium (Corning) supplemented with 10% FBS, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 2 mM of L-glutamine at 37°C/5% CO2.

Zanoni M., Palesch D., Pinacchio C., Statzu M., Tharp G.K., Paiardini M., Chahroudi A., Bosinger S.E., Yoon J., Cox B., Silvestri G, & Kulpa D.A. (2020). Innate, non-cytolytic CD8+ T cell-mediated suppression of HIV replication by MHC-independent inhibition of virus transcription. PLoS Pathogens, 16(9), e1008821.

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