Il 15

Manufactured by CellGenix

Sourced in Germany

IL-15 is a recombinant cytokine produced by CellGenix. It is a member of the common gamma-chain cytokine family and plays a crucial role in the activation and proliferation of natural killer cells and T cells.

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Lab products found in correlation

Interleukin 7 (il 7), by CellGenix (5 mentions) Lymphoprep, by STEMCELL (2 mentions) Macs human nk cell isolation kit, by Miltenyi Biotec (2 mentions) Gm csf, by CellGenix (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Interleukin 2 (il 2), by Miltenyi Biotec (2 mentions) Control igg, by R&D Systems (1 mentions) Cytofix, by BD (1 mentions) Anti il 15rα, by R&D Systems (1 mentions) Alexa fluor 647, by BD (1 mentions) Il 12p70, by R&D Systems (1 mentions) 7 aminoactinomycin d, by BD (1 mentions) Easysep human t cell isolation kit, by STEMCELL (1 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions) Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions) Click s, by Irvine Scientific (1 mentions) Interleukin 7 (il 7), by R&D Systems (1 mentions) Il 12, by R&D Systems (1 mentions) Cd16 apc, by Miltenyi Biotec (1 mentions) Cd56 apc vio770, by Miltenyi Biotec (1 mentions)

Spelling variants of this product

Recombinant human IL-15 (5 citations)

15 protocols using il 15

Characterizing STAT Signaling in Immune Cells

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STAT5 signaling: PBMCs (1 × 10⁶ cells /mL) were stimulated overnight with IL15 (7.5 ng/mL; CellGenix)(21 (link)), harvested, then serum-starved for 1hr in the presence of TG101348, ruxolitinib, or DMSO control. The cells were then pulsed with IL2 (EC₅₀ 50 IU/mL; Chiron), IL15 (EC₅₀ 2.7 ng/mL; CellGenix), allogeneic moDCs or LCs (1:1 ratio) for 15 minutes followed by fixation (CytoFix; BD Biosciences)and permeabilization. Cells were stained for CD3 (Clone UCHT1) and CD56 to identify NK cells (CD3⁻CD56⁺) and intracellular Alexa Fluor 647 anti-STAT5 (pY694; BD Biosciences). Where indicated, IL15Rα was blocked on both moDCs and LCs with anti-IL15Rα(5μg/mL)or IgG control (R&D Systems).
STAT4 signaling: PBMCs (1 × 10⁶ cells /mL) were stimulated for 2 d with 100 IU/mL IL2 (Chiron) to facilitate optimal IL12R induction(22 (link)). Cells were harvested and serum-starved for 1 hour in the presence of TG101348, ruxolitinib, or DMSO. Cells were then pulsed with allogeneic moDCs or LCs (1:1 ratio), matured moDC or LC supernatant, or 100 IU/mL IL12p70 (R&D Systems) for 90 min (23 (link)). pSTAT4 expression was ascertained using an intracellular PE- or Alexa Fluor 647-conjugated anti-STAT4 (pY693; BD Biosceinces).
STAT3 signaling: This was assessed in JAK inhibitor-treated T cells as published(3 (link)).

Curran S.A., Shyer J.A., St. Angelo E.T., Talbot L.R., Sharma S., Chung D.J., Heller G., Hsu K.C., Betts B.C, & Young J.W. (2016). Human Dendritic Cells Mitigate NK-Cell Dysfunction Mediated by Nonselective JAK1/2 Blockade. Cancer immunology research, 5(1), 52-60.

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T Cell Activation and Cryopreservation

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CD3⁺ T cells were purified from healthy human donor blood preparations by negative magnetic bead isolation (EasySep^TM Human T Cell Isolation Kit, Stemcell Technologies) and cryopreserved. Thawed cells were activated using Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher) at a ratio of 1:1 bead:cell in test media or reference media in the presence of IL-7 (10 ng/ml, CellGenix) and IL-15 (10 ng/ml, CellGenix). Cells were cultured at 37 °C in a humidified incubator at 5% CO₂ in 96-well U bottom plates with two to three repeats per condition. On day 3 cells were splitted and reseeded with cytokine-containing media. To determine cell viability, cells were labeled with 7-Amino-Actinomycin D (7-AAD, BD-Pharmingen) and analyzed by flow cytometry. Cell count was determined using an Attune Nxt Flow Cytometer (Thermo Fisher).

Grzesik P, & Warth S.C. (2021). One-Time Optimization of Advanced T Cell Culture Media Using a Machine Learning Pipeline. Frontiers in Bioengineering and Biotechnology, 9, 614324.

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Generating Antigen-Specific CD8+ T Cells

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PBMCs were isolated using Ficoll as described above. Adherent cells were used for DC generation, and T-cell-containing nonadherent cells were frozen for use on day 7. After thawing, nonadherent cells were labeled for immunomagnetic selection of CD45RA+ cells, washed, and then selected by MACS^®. The CD45RA+ cells were then resuspended in 45% RPMI (Hyclone) and 45% CLICKS (Irvine Scientific) with 10% Human Serum plus GlutaMAX^™ (T-cell medium). Cells were resuspended at 2×10⁶/ml and cocultured with autologous, Pepmix-pulsed DCs at a ratio of 20 PBMCs to 1 DC in the presence of the cytokines 10 ng/ml IL-7 and IL-12, (R&D Systems) and 5 ng/mL IL-15 (CellGenix). Cultures were restimulated on days 10 and 17 with irradiated (40 Gy), pp65 Pepmix-pulsed autologous LCLs at a responder-to-stimulator ratio of 4:1 plus IL-15 (5 ng/ml) on day 10 and 50 U/mL IL-2 (Proleukin) on days 17 and day 20. To confirm the origin of the pp65-specific T-cell populations we sorted CD45RA/CCR7 double positive and double negative T-cell populations by flow cytometry and stimulated them with pp65-Pepmix-pulsed DCs followed by pp65-pepmix-pulsed LCLs as described above.

Hanley P.J., Melenhorst J.J., Nikiforow S., Scheinberg P., Blaney J.W., Demmler-Harrison G., Cruz C.R., Lam S., Krance R.A., Leung K.S., Martinez C.A., Liu H., Heslop H.E., Rooney C.M., Shpall E.J., Barrett A.J., Rodgers J.R, & Bollard C.M. (2015). CMV-Specific T-cells Generated From Naïve T-cells Recognize Atypical Epitopes And May Be Protective in Vivo. Science translational medicine, 7(285), 285ra63.

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Isolation and Activation of Human NK Cells

Cited 1 time

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Mononuclear cells (MNCs) were isolated from peripheral blood using Lymphoprep™ (STEMCELL Technologies, The Netherlands) density gradient centrifugation from buffy coats obtained from anonymous healthy blood donors (Sanquin Blood Supply, Amsterdam) with written informed consent for research use, in accordance with the ‘‘Code for Proper Use of Human Tissues” as formulated by the Dutch Federation of Medical Scientific Organizations (www.fmwv.nl) [44 (link)]. CD56⁺ NK cells were isolated from MNCs using a MACS Human NK cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The cell number and purity of the isolated NK cell fraction were analyzed by flow cytometry. Isolated NK cells were activated overnight with 1000U/ml IL-2 (Proleukin^®; Chiron, München, Germany) and 10ng/ml IL-15 (CellGenix) for use in cytotoxicity assays. NK cell purity and viability were checked using CD3 PE, 7AAD (BD Biosciences), CD56 APC Vio 770, and CD16 APC (Miltenyi Biotech). The parameters compared before and after activation were NK purity (CD56⁺%, 83 ± 9% vs. 82 ± 9%), NK CD16% 88 ± 10% vs 85 ± 11%) and NK viability (91 ± 3% vs 86 ± 2%) respectively.

Veluchamy J.P., Spanholtz J., Tordoir M., Thijssen V.L., Heideman D.A., Verheul H.M., de Gruijl T.D, & van der Vliet H.J. (2016). Combination of NK Cells and Cetuximab to Enhance Anti-Tumor Responses in RAS Mutant Metastatic Colorectal Cancer. PLoS ONE, 11(6), e0157830.

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Expanding CD25+ CMV-Specific T Cells

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After isolation of CD25+ CMV-T, up to 0.25×10⁶ cells were cultured in the presence of 12.5×10⁶ (50∶1) γ-irradiated (30 Gy) autologous PBMCs to act as feeder cells in 24 well plates with RPMI 1640 medium containing 10% human AB serum, 1% antibiotic and supplemented with 10 ng/ml of IL-7 and IL-15 (CellGenix, Freiburg, Germany). Culture medium was replenished every 2–3 days and cells split when necessary. Cells were expanded up to a maximum of 24 days before harvest.

Samuel E.R., Beloki L., Newton K., Mackinnon S, & Lowdell M.W. (2014). Isolation of Highly Suppressive CD25+FoxP3+ T Regulatory Cells from G-CSF-Mobilized Donors with Retention of Cytotoxic Anti-Viral CTLs: Application for Multi-Functional Immunotherapy Post Stem Cell Transplantation. PLoS ONE, 9(1), e85911.

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Isolation and Activation of NK Cells

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Whole blood samples from four healthy volunteers were collected. PBMC were isolated using Lymphoprep™ (STEMCELL Technologies, The Netherlands) density gradient centrifugation. CD56⁺ NK cells were isolated from PBMC using a MACS^® Human NK cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The cell number and purity of the isolated PBNK was analyzed by flow cytometry. Isolated NK cells were activated overnight with 1000 U/mL IL-2 (Proleukin^®; Chiron, München, Germany) and 10 ng/mL IL-15 (CellGenix) before use in cytotoxicity assays. NK cell purity and viability were checked by flow cytometry using the following antibodies: 7-aminoactinomycin D (7AAD; Sigma-Aldrich), CD3 (labeled with VioBlue), CD56 (labeled with APC-Vio770) and CD16 (labeled with APC) (all from Miltenyi Biotech). Purity of NK cells obtained from NK donors was 87 ± 6 %. For cytotoxicity assays, only PBNK with CD16 expression rates exceeding 80 % were used.

Veluchamy J.P., Heeren A.M., Spanholtz J., van Eendenburg J.D., Heideman D.A., Kenter G.G., Verheul H.M., van der Vliet H.J., Jordanova E.S, & de Gruijl T.D. (2016). High-efficiency lysis of cervical cancer by allogeneic NK cells derived from umbilical cord progenitors is independent of HLA status. Cancer Immunology, Immunotherapy, 66(1), 51-61.

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Activation of Antigen-Specific T Cells

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PBMCs were seeded to culture plates in T cell media supplemented with dendritic cell supporting factors GM-CSF at 1000U/mL (CellGenix, #1412–050) and IL-4 at 500 U/mL (CellGenix, #1403–050), T cell supporting cytokines IL-7 at 5 ng/mL (CellGenix, # 1410–050), IL-15 at 5 ng/ml (CellGenix #1413–050), and IL-2 at 10 U/ml (Peprotech, #200–02). Individual peptides or pools of peptides were added to cells at 10 µg/ml (Genscript). Medium containing cytokines was replenished every 2 to 3 days. Cells were re-stimulated with the individual peptides or peptide pools they were initially stimulated with or re-stimulated with DMSO, a control CEF peptide pool or anti-CD3 (clone HIT3a)/anti-CD28 (clone CD28.2) and analyzed 24 h later by flow cytometry for activation induced markers. The antibodies used for flow cytometry analysis were described in Antigen-specific T cell reactivity assays.

Deering R.P., Blumenberg L., Li L., Dhanik A., Jeong S., Pourpe S., Song H., Boucher L., Ragunathan S., Li Y., Zhong M., Kuhnert J., Adler C., Hawkins P., Gupta N.T., Moore M., Ni M., Hansen J., Wei Y, & Thurston G. (2023). Rapid TCR:Epitope Ranker (RAPTER): a primary human T cell reactivity screening assay pairing epitope and TCR at single cell resolution. Scientific Reports, 13, 8452.

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Polyclonal T cell Activation Protocol

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T cells were cultured in T cell medium containing RPMI 1640 (Gibco), 10% heat-inactivated fetal calf serum (FCS) (Sigma-Aldrich), recombinant IL-7 (10 ng/mL, Cell-Genix), and IL-15 (5 ng/mL, Cell-Genix). All cell culture experiments were performed at 37 °C and 5% CO₂. Polyclonal T cell stimulation was performed for 48 h on anti-CD3/anti-CD28-coated tissue culture plates. Coating of vacuum gas plasma-treated polystyrene 24-Well-Tissue-Culture plates (Corning) was performed overnight with 500 μL/well of sterile ddH2O (Ampuwa) supplemented with 1 μg/mL anti-CD3 monoclonal antibody (mAb) (clone OKT3; Invitrogen) and 1 μg/mL anti-CD28 mAb (clone CD28.2; BioLegend). Plates were washed twice in PBS and once in RPMI without letting the wells dry out. T cells were seeded at a density of 1–1.5 × 10⁶ per well in a 24-well plate. For allogeneic T cell generation, PBMCs were depleted of NK cells using LD columns and the NK cell isolation kit, human (Miltenyi Biotec, Germany). Subsequently, allo-reactive T cells were stimulated 1:1 by adding irradiated CD3-depleted PBMCs from a different donor at the day of isolation. Allo-specific T cells were re-stimulated in a 1:1 ratio with the same CD3-depleted PBMCs on day 5 after isolation and expansion.

Glaser V., Flugel C., Kath J., Du W., Drosdek V., Franke C., Stein M., Pruß A., Schmueck-Henneresse M., Volk H.D., Reinke P, & Wagner D.L. (2023). Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells. Genome Biology, 24, 89.

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Expansion of Antigen-Specific T Cells

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For short-term expansion (STE), 2 x 10⁷ PBMCs were cultured in G-REX devices (Wilson Wolf Manufacturing, New Brighton, MN) in 2 ml AIM-V medium (Invitrogen, Carlsbad, CA) supplemented with 25 mmol/l L-Glutamin (ThermoFisher Scientific, Waltham. MA), 25 mmol/l HEPES (ThermoFisher Scientific) and 2% Octaplas AB (Octapharma, Vienna, Austria) (named AIMV^{+ + +}, as already described (19 (link))) and stimulated for 12 days with either the Asp-lysate (50 µg/ml) alone or with combined overlapping peptide pools (referred to as PepMix) from Asp and Candida albicans (CatB1, Crf1, f22, Gel1, pmp20, SHMT, SOD, MP65; all from Miltenyi Biotec; 0.6 nmol/ml). On day six, cells were washed with AIMV^{+ + +} and resuspended and restimulated as on day 0. On day nine, IL-15 (CellGenix, Freiburg, Germany, 5 ng/ml) was added to the cell culture and seATCs were characterized by multi-parameter flow cytometry and IFN-γ EliSpot on day 12.

Tischer-Zimmermann S., Salzer E., Bitencourt T., Frank N., Hoffmann-Freimüller C., Stemberger J., Maecker-Kolhoff B., Blasczyk R., Witt V., Fritsch G., Paster W., Lion T., Eiz-Vesper B, & Geyeregger R. (2023). Rapid and sustained T cell-based immunotherapy against invasive fungal disease via a combined two step procedure. Frontiers in Immunology, 14, 988947.

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Tumor-Infiltrating Lymphocyte Expansion

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TILs were expanded from patient tumor tissue and cell culture experiments performed with TILGen subject samples to determine neoepitope recognition. Biopsies obtained from the two TILGen patients were cultured in Roswell Park Memorial Institute (RPMI) with 40 U/mL penicillin/streptomycin, 2 mM L-glutamine, 50 µM β-mercaptoethanol (all Gibco), and 5% fetal calf serum (FCS), 5% human serum, 0.4% vitamin solution, 1% minimal essential medium, and 1 mM sodium pyruvate (all PAN-Biotech, Germany) in the presence of 2.5×10⁵ CD3/CD28 beads/mL (Gibco), 100 U/mL IL-2 (Proleukin) and 5 ng/mL IL-15 (Cellgenix, Germany) for 14–21 days.

Reimann H., Nguyen A., Sanborn J.Z., Vaske C.J., Benz S.C., Niazi K., Rabizadeh S., Spilman P., Mackensen A., Ruebner M., Hein A., Beckmann M.W., van der Meijden E.D., Bausenwein J., Kretschmann S., Griffioen M., Schlom J., Gulley J.L., Lee K.L., Hamilton D.H., Soon-Shiong P., Fasching P.A, & Kremer A.N. (2021). Identification and validation of expressed HLA-binding breast cancer neoepitopes for potential use in individualized cancer therapy. Journal for Immunotherapy of Cancer, 9(6), e002605.

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