Il 15

Manufactured by Miltenyi Biotec

Sourced in Germany, United States, United Kingdom, Japan, Switzerland

IL-15 is a recombinant human cytokine that can be used as a cell culture supplement. It functions to stimulate the proliferation and activation of T cells and natural killer cells.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

Human IL-15 (17 citations) Recombinant human IL-15 (10 citations) IL-7 and IL-15 (9 citations) Recombinant human IL-7 and IL-15 (3 citations) MACS GMP Recombinant Human IL-15 (2 citations)

145 protocols using il 15

T Cell Transduction with RetroCAR

Check if the same lab product or an alternative is used in the 5 most similar protocols

Isolated TRBC1⁺ or TRBC2⁺ T cells were resuspended at 1 × 10⁶ cells/mL in R10 and stimulated with TransAct (Miltenyi Biotec; 130-111-160), 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). 24 h after, cells were collected, plated at a density of 1 × 10⁶ cells per well (1 mL) on retronectin-coated (Takara, T100B) 6-well plates in the presence of retroviral supernatant at an MOI of 1. Total volume was adjusted to 3 mL using R10 supplemented with 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). The plate were centrifuged at 1000 g for 40 min. 24 h post spinoculation, the T cells were harvested and re-plated in complete R10 media supplemented with 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). Transduction efficiency was determined on day 5 after transduction, and further experiments were commenced on days 5–9 after transduction. CAR expression was assessed by staining with aCD3-PE/Cy7 (317334, Biolegend) and QBend10 APC (FAB7227A, R&D System).

Ferrari M., Righi M., Baldan V., Wawrzyniecka P., Bulek A., Kinna A., Ma B., Bughda R., Akbar Z., Srivastava S., Gannon I., Robson M., Sillibourne J., Jha R., El-Kholy M., Amin O.M., Kokalaki E., Banani M.A., Hussain R., Day W., Lim W.C., Ghongane P., Hopkins J.R., Jungherz D., Herling M., Welin M., Surade S., Dyson M., McCafferty J., Logan D., Cordoba S., Thomas S., Sewell A., Maciocia P., Onuoha S, & Pule M. (2024). Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies. Nature Communications, 15, 1583.

+ Open protocol

+ Expand

Isolation and Transduction of T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

CD4⁺, CD8⁺, or mixed CD4⁺ and CD8⁺ T cells were isolated with the RosetteSep CD4⁺ or CD8⁺ Human T-cell Enrichment Cocktail (Stemcell Technologies) from healthy donor whole blood obtained from the UCLA Blood and Platelet Center. T-cells stimulated with CD3/CD28 Dynabeads (Thermo Fisher Scientific) at a 1:1 cell:bead ratio for 2 days were transduced with lentivirus at a multiplicity of infection of 1.5–3. T cells were cultured in complete RPMI and fed 50 U/ml IL-2 (Thermo Fisher Scientific) and 1 ng/ml IL-15 (Miltenyi Biotec) every 2 to 3 days. Dynabeads were removed after 9 days of culture. Transduced cells were enriched by magnetic bead-based sorting (Miltenyi Biotec) and continued to be cultured with IL-2/IL-15 supplementation every 2 to 3 days.

Chang Z.L., Lorenzini M.H., Chen X., Tran U., Bangayan N.J, & Chen Y.Y. (2018). Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nature chemical biology, 14(3), 317-324.

+ Open protocol

+ Expand

Isolation and Transduction of T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Generating CAR-T Cells from PBMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Peripheral blood mononuclear cells (PBMC) were isolated from volunteer healthy donors’ peripheral blood (PB) or buffy coats by density-gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway) or Ficoll Paque Plus (GE Healthcare, Little Chalfont, UK). PBMC were plate-coated activated with 1 µg/mL anti-CD3 (OKT3) and 1 µg/mL anti-CD28 (CD28.2) mAbs (BD Biosciences) for 2 days and transduced (MOI of 10) with CD1a-CAR- or CD1a-STAb-encoding lentiviruses in the presence of 10 ng/mL interleukin (IL)-7 and 10 ng/mL IL-15 (Miltenyi Biotec, Bergisch Gladbach, Germany). As negative controls, non-transduced or GFP-transduced T cells were used (NT). T cells were expanded in RCM supplemented with IL-7 and IL-15 (10 ng/mL) (Miltenyi Biotec).22 32 (link)

Jiménez-Reinoso A., Tirado N., Martinez-Moreno A., Díaz V.M., García-Peydró M., Hangiu O., Díez-Alonso L., Albitre Á., Penela P., Toribio M.L., Menéndez P., Álvarez-Vallina L, & Sánchez Martínez D. (2022). Efficient preclinical treatment of cortical T cell acute lymphoblastic leukemia with T lymphocytes secreting anti-CD1a T cell engagers. Journal for Immunotherapy of Cancer, 10(12), e005333.

+ Open protocol

+ Expand

Cell Culture and PBMC Isolation

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK-293T and HT1080 cells were cultivated in DMEM (Biowest, Nuaille, France) (10% FCS (Biochrom, Berlin, Germany)). Raji and SupT1 cells were cultured in RPMI (Biowest, Nuaille, France) supplemented with 10% FCS and 2 mM L-glutamine (Biowest, Nuaille, France). PBMCs were isolated from buffy coat derived from healthy donors via density centrifugation using Ficoll (Pan Biotech, Aidenbach, Germany) and cultured in TexMACS™ (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 12.5 ng/mL IL-7 and 12.5 ng/mL IL-15 (both Miltenyi Biotec, Bergisch Gladbach, Germany). For the selectivity assay, PBMCs were cultivated in TexMACS™ supplemented with 50 IU/mL IL-4, 12.5 ng/mL IL-7, and 12.5 ng/mL IL-15 (all Miltenyi Biotec, Bergisch Gladbach, Germany) and 1 µg/mL CD40-ligand (RD Systems, Minneapolis, MN, USA).

Cordes N., Winter N., Kolbe C., Kotter B., Mittelstaet J., Assenmacher M., Cathomen T., Kaiser A, & Schaser T. (2022). Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer. Viruses, 14(10), 2157.

+ Open protocol

+ Expand

Verification of Peptide-Specific CTL

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 5

To confirm induction of PSF1 peptide specific CTL with another method, a Tetramer assay was conducted.

(Method)

Production of CTL Line

Cells that had been determined to be positive in the above-described ELISPOT assay was continued to be incubated with a culture solution in which 100 ng/ml of IL-15 (Miltenyi Biotec K.K., San Diego, Calif.) was added to an AIM-V culture medium including antibiotic and serum.

Tetramer Assay

Tetramer (Medical & Biological Laboratories Co., Ltd: MBL) for PSF1 peptides (YLYDRLLR, SEQ ID NO.: 31) was produced. Cells of a CTL line were collected, colored with tetramer and APC H7 marker anti-CD8 antibodies (SK-1) (Becton, Dickinson and Company, La Jolla, Calif.), and measured with a FACS Aria (Becton, Dickinson and CompanyLa Jolla, Calif.). Two types of CTL lines (0209-01 H2 and 0209-02 D2) were used.

(Results)

As shown in FIG. 1, since a cell population that reacts with both anti-CD8 antibodies and tetramer was observed in each CTL line, it was found that CTL lines recognizing PSF1 peptides were produced. Observation of Tetramer-positive cells directly proved the presence of peptide-specific human CTL.

US10617750B2. Partner of SLD five 1 (PSF1)-derived peptide (2020-04-14). Vasculead Inc. [JP]. Inventors: Hidekazu Tanaka [JP], Motofumi Iguchi [JP], Mari Yokoyama [JP].

+ Open protocol

+ Expand

Isolation and Transduction of Primary T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For in vitro assays, buffy coats from healthy donors were obtained from DRK Dortmund. Density gradient centrifugation with Pancoll human (PAN-Biotech, catalog no.: P04-601000) was used for PBMC isolation. T cells were isolated from PBMCs with the Pan T Cell Isolation Kit, human (Miltenyi Biotec, catalog no.: 130-096-535). Isolated T cells were cultured in TexMACS™ Medium (Miltenyi Biotec, catalog no.: 130-097-196) supplemented with 12.5 ng/mL IL-7 (Miltenyi Biotec, catalog no.: 170-076-111) and 12.5 ng/mL IL-15 (Miltenyi Biotec, catalog no.: 170-076-114). For activation before transduction, T cells were seeded in a 24-well plate at a density of 1 × 10⁶ cells/mL and T Cell TransAct™ (Miltenyi Biotec, catalog no.: 130-111-160) was added. After 24 h, the cells were transduced with an MOI of 10. After 3 d, the culture medium was exchanged completely to remove residual LVV particles. T cells were cultured for 10–14 d with the regular addition of fresh culture medium every 2 d. CAR T cells for in vivo studies were manufactured using the CliniMACS Prodigy™ (Miltenyi Biotec) as described previously by Lock et al.^{30 (link)} As starting material leukapheresis obtained from “Institut für klinische Transfusionsmedizin und Immungenetik Ulm” was used. The enriched T cells were transduced at an MOI of 10 and cultured until day 10 for harvesting.

Werchau N., Kotter B., Criado-Moronati E., Gosselink A., Cordes N., Lock D., Lennartz S., Kolbe C., Winter N., Teppert K., Engert F., Webster B., Mittelstaet J., Schaefer D., Mallmann P., Mallmann M.R., Ratiu D., Assenmacher M., Schaser T., von Bergwelt-Baildon M., Abramowski P, & Kaiser A.D. (2022). Combined targeting of soluble latent TGF-ß and a solid tumor-associated antigen with adapter CAR T cells. Oncoimmunology, 11(1), 2140534.

+ Open protocol

+ Expand

Isolation and Stimulation of CD4+ T-cells and NK-cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

CD4⁺T-cells were isolated from frozen PBMCs. All CD4⁺T-cells were positively selected with a CD4⁺T-cell isolation kit (Miltenyi Biotec, Germany), yielding CD4⁺T-cell populations at a purity of 96–99%. Purified CD4⁺T-cells were stimulated as described previously (28 (link)) with 4 μg/mL plate-bound anti-human CD3 (OKT3) mAb (eBioscience, San Diego, CA) and 4 μg/mL soluble anti-human CD28 (CD28.2) mAb (Becton Dickinson) in presence of Recombinant human IL-2 (Proleukine, Chiron, Amsterdam, 100 U/mL) and recombinant human interferon alpha-2a (Roferon-A) and IFNλ2 (Biotechne, UK) at the indicated dose. After five days culture, CD38 and CD25 expression as well as the frequency of 7AAD⁺ cells were measured by flow cytometry on stimulated CD4⁺T-cells (Table S4). NK-cells were isolated from PBMCs. NK-cells were negatively selected with the NK-cell isolation kit (Miltenyi Biotec, Germany), yielding NK-cell populations at a purity of 96–99%. NK-cells were stimulated with IL-15 (Miltenyi Biotec, 10 ng/mL), IL-2 (Proleukine, Chiron, Amsterdam 100 U/mL) and recombinant human interferon alpha-2a (Roferon-A) at the indicated dose. After 3 days of culture, expression of CD56, CD95 and NKG2D was measured by flow cytometry (extended data Table 3a).

Le Buanec H., Schiavon V., Merandet M., How-Kit A., Bergerat D., Fombellida-Lopez C., Bensussan A., Bouaziz J.D., Burny A., Darcis G., Song H., Sajadi M.M., Kottilil S., Gallo R.C, & Zagury D. (2023). Early Elevated IFNα Identified as the Key Mediator of HIV Pathogenesis and its low level a Hallmark of Elite Controllers. Research Square.

+ Open protocol

+ Expand

NK Cell Cytotoxicity Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs, 5 × 10 5 , were incubated overnight in 0•5 ml RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin with or without 1 ng/ ml IL-15 (Miltenyi Biotec, Auburn, CA, USA) at 37°C, 5% CO 2 . Then, PBMCs were incubated for 1 h at 37°C with mouse anti-human CD107a APC-conjugate antibody (clone H4A3; Miltenyi Biotec) in contact with K562 target cells, a human erythroleukemic cell line which does not express major histocompatibility complex (MHC) class I molecules (effector to target ratio 5 : 1). Then, 10 μg/ml brefeldin A (Sigma-Aldrich) and 6 μg/ml monensin (Sigma-Aldrich) were added to the cells for an additional 3 h at 37°C, 5% CO 2 . Cells were stained with live/dead fixable dead cell stain near-IR-fluorescent reactive dye (Life Technologies, Carlsbad, CA, USA) for 15 min at room temperature, followed by staining with CD3 and CD56 antibody for 25 min at 4°C. FACS Canto II flow cytometer (BD Biosciences) and FACSDiva version 8.0.1 software were used for the acquisition and data analysis, respectively.

Bonacini M., Cimino L., De Simone L., Bolletta E., Gozzi F., Soriano A., Muratore F., Zerbini A., Fontana L., Salvarani C, & Croci S. (2021). Vogt-Koyanagi-Harada patients show higher frequencies of circulating NKG2D^pos NK and NK T cells. Clinical and experimental immunology, 204(1).

+ Open protocol

+ Expand

Matrix-Free Patient-Derived Tumoroids

Check if the same lab product or an alternative is used in the 5 most similar protocols

As described before in our previous studies, for the formation of patient-derived tumoroids (PDTs) and healthy organoids (PDHOs) in a matrix-free condition, supportive cells such as adipose-tissue-derived microvessels (ad-MVs) were added (12 (link)). They were prepared according to the protocol from the supplier Advanced Solutions® and the previous studies.
PDTs and PDHOs were prepared according to a mix of 5000 patients’ cells and 5000 ad-MVs (within 1 PDT or PDHO). Cells’ suspension was then diluted in cell culture media which is composed of a mix of DMEM high glucose (ref. 41966-029, Gibco), RPMI (ref. 10101-145, Sigma) and TexMACS™ media (ref. 130-097-196, Miltenyi Biotech). This cell culture media was supplemented with different growth factors to improve patient ‘s cells in vitro culture, maintenance of tumor-infiltrating immune cells and ad-MVs (details in Table 1).
After that, 200µL of this cell suspension was transferred on a ULA U bottom 96 wells plate (ref. 174929, Thermofisher) to form PDTs and PDHOs. The medium was changed every 3-4 days by replacing IL-2 with two other interleukins: IL-7 at 155UI/mL (ref. 130-095-361, Miltenyi Biotech) and IL-15 at 290UI/mL (ref. 130-095-762, Miltenyi Biotech).

Lê H., Deforges J., Cutolo P., Lamarque A., Hua G., Lindner V., Jain S., Balloul J.M., Benkirane-Jessel N, & Quéméneur E. (2024). Patient-derived tumoroids and proteomic signatures: tools for early drug discovery. Frontiers in Immunology, 15, 1379613.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!