Human Interleukin-15 Recombinant

HomeHematopoietins, IL-2 family, Recombinant Human Cytokines

Human Interleukin-15 Recombinant

$70.00 – $3,500.00

SKU: RKP40933 Categories: Hematopoietins, IL-2 family, Recombinant Human Cytokines Tags: il-15, il15, Interleukin-15, P40933

Description

Methods (21)


accession	P40933

Source	Optimized DNA sequence encoding Human Interleukin-15 mature chain was expressed in Escherichia Coli.
Molecular weight	Native human Interleukin-15 is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately 13 kDa. Recombinant Interleukin-15 is a monomer protein consisting of 115 amino acid residue subunits, and migrates as an approximately 13kDa protein under reducing conditions in SDS-PAGE.
Purity	>96%, as determined by SDS-PAGE and HPLC
Biological Activity	The ED(50) was determined by the dose-dependent proliferation ofmouseCTLL-2 cells and was found less than0.3 ng/ml.
Protein Sequence	MRISKPHLRS ISIQCYLCLL LNSHFLTEAG IHVFILGCFS AGLPKTEANW VNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS
Endotoxin	Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation	Recombinant IL-15 was lyophilized from a.2 μm filtered PBS solution pH7.5 .
Reconstitution	A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers.
Storage	The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage	This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.

Interactor	P14784 IL2RB_HUMAN
Interactor	P31785 IL2RG_HUMAN
Interactor	P41273 TNFL9_HUMAN
Molecular function	Cytokine

Methods


Isolation of MiHA specific T-cells by pMHC tetramer pull down Prior to isolation of peptide-specific T-cells, pMHC tetramers were made coupled to SA-PE. PBMC were stained with pMHC tetramers for 1 hour at 4°C. Subsequently, cells were washed and incubated with anti-PE Ab coated magnetic beads . Cells were than isolated by MACS , using an LS column, following the manufacturers protocol. Eluted cells were washed and cultured in Iscove Modified Dulbecco Medium (IMDM; ) supplemented with 5% human serum, 5% fetal calf serum (FCS), 100 IU/mL IL-2 , 10ng/mL IL-15 . Eluted cells were cultured per 5000 cells with 2×10⁴ irradiated autologous feeder cells and 5000 anti-CD3/CD28 Dynabeads in 96-well plates. Cultures were split at least twice a week. After 2–3 weeks, cell cultures were analyzed for peptide-specific T-cell populations by pMHC tetramer flow cytometry. Subsequently pMHC tetramer reactive T-cell populations were sorted on a FACSAria into 96 well plates containing 1×10⁵ irradiated feeder cells supplemented… Prior to isolation of peptide-specific T-cells, pMHC tetramers were made coupled to SA-PE. PBMC were stained with pMHC tetramers for 1 hour at 4°C. Subsequently, cells were washed and incubated with anti-PE Ab coated magnetic beads . Cells were than isolated by MACS , using an LS column, following the manufacturers protocol. Eluted cells were washed and cultured in Iscove Modified Dulbecco Medium (IMDM; ) supplemented with 5% human serum, 5% fetal calf serum (FCS), 100 IU/mL IL-2 , 10ng/mL IL-15 . Eluted cells were cultured per 5000 cells with 2×10⁴ irradiated autologous feeder cells and 5000 anti-CD3/CD28 Dynabeads in 96-well plates. Cultures were split at least twice a week. After 2–3 weeks, cell cultures were analyzed for peptide-specific T-cell populations by pMHC tetramer flow cytometry. Subsequently pMHC tetramer reactive T-cell populations were sorted on a FACSAria into 96 well plates containing 1×10⁵ irradiated feeder cells supplemented with 0.5 µg/mL phytohaemagglutinin (PHA ) as previously described Read more
Cell isolation and culture Peripheral blood mononuclear cells (PBMC) from CLL patients or healthy donors were separated from heparinized venous blood by Ficoll density gradient centrifugation. The PBMC used in this study were generally greater than 90% CLL B cells as determined by CD19/CD5 positivity. Leukemic cells were cultured in serum-free adoptive immunotherapy media-V medium'>(AIM-V) medium . For CLL B cell activation experiments, cells were cultured for 5 days in AIM-V media or AIM-V media supplemented with 2.5 µg/ml CpG ODN 2006 (provided by core Mayo Clinic facility) with or without 94 IU/ml human IL-2 , 10 ng/ml human IL-15 , and 13 µM beta-mercaptoethanol. Further CLL B cell activation experiments utilized 12-O-tetradecanoylphorbol-13-acetate (TPA) at 10 ng/ml or interferon-α 2b at 1000 IU/ml. Human C4+T cells were isolated from healthy donor PBMC by immunomagnetic selection using T cell enrichment kits and incubated with CLL B cells… Peripheral blood mononuclear cells (PBMC) from CLL patients or healthy donors were separated from heparinized venous blood by Ficoll density gradient centrifugation. The PBMC used in this study were generally greater than 90% CLL B cells as determined by CD19/CD5 positivity. Leukemic cells were cultured in serum-free adoptive immunotherapy media-V medium'>(AIM-V) medium . For CLL B cell activation experiments, cells were cultured for 5 days in AIM-V media or AIM-V media supplemented with 2.5 µg/ml CpG ODN 2006 (provided by core Mayo Clinic facility) with or without 94 IU/ml human IL-2 , 10 ng/ml human IL-15 , and 13 µM beta-mercaptoethanol. Further CLL B cell activation experiments utilized 12-O-tetradecanoylphorbol-13-acetate (TPA) at 10 ng/ml or interferon-α 2b at 1000 IU/ml. Human C4+T cells were isolated from healthy donor PBMC by immunomagnetic selection using T cell enrichment kits and incubated with CLL B cells at a ratio of 1∶2 in plates pre-coated with PBS containing 10 µg/ml of anti-C3 and anti-C28 (B) stimulating antibodies. All cells were maintained in appropriate media at 37°C in an atmosphere containing 95% air and 5% CO₂. Read more
PAF-stimulated Th17 development is dependent on cytokines, LC-T cell contact and selected signaling pathways. (a) Monocyte-derived LC were stimulated with either vehicle or PAF in the presence of neutralizing Ab to IL-15, IL-6R, and/or IL-23, or control Ig, and cocultured with antiCD3/CD28-activated CD4+ T cells for 5 days.
Donor APCs plus γc cytokines induce rapid expansion of CD8+CD28− T cells in culture. Freshly purified CD8+ T cells (2×106 per well) from healthy volunteers were stimulated by HLA-A, B and DR mismatched allogeneic APCs (1×106 per well) for 9 days in 24-well plates supplemented with IL-2 alone or a combination of IL-2, IL-7 and IL-15.
Human in vitro iNKT cell assay Human iNKT cell clones have been previously described. Human primary iNKT cell lines were generated by expanding freshly isolated PBMC in 50 U/ml IL-2 and 5 ng/ml IL-15 in culture with 10 ng/ml α-GalCer for 14 days. iNKT cells were purified by PBS-57-loaded CD1d tetramer selection with magnetic beads , and were > 99% CD3⁺PBS-57-tet⁺. Generation of human PBMC-derived monocytes has been previously described. 5 × 10⁴ iNKT cells were cultured with 5 × 10⁴ PBMC-derived monocytes per well.
The co-culture model system The in vitro model simulating the human tumor microenvironment contained 5 × 10⁵ iDC co-cultured with 5 × 10⁵ irradiated (3,000 rad) PCI-13 cells and autologous CD4⁺CD25⁻ T cells (5 × 10⁶) in six-well plates [₂ in air at 37°C for 10 days. In addition, 2.5 mL aliquots of IRX-2 or X-Vivo 10 medium (control) were added to each well as well as rhIL-2 (10 IU/mL), IL-10 (20 IU/mL), and IL-15 (20 IU/mL) . On days 3, 6, and 9, half of the medium was removed and replaced with fresh cytokine-containing medium mixed 1:1 with medium or IRX-2. For cytokine assays, cells were stimulated for 16 h with anti-CD3/CD28-coated beads at the bead:cell ratio of 1:1 in complete medium'>AIM V medium without exogenous cytokines or IRX-2. For intracellular cytokine staining, Brefeldin A (2 μg/mL, , ) was added to the cells.
CD56bright NK cells have a higher cytotoxic potential towards activated CD4+ T cells. NK cells were activated as indicated: 200 IU/mL IL-2, 25 ng/mL IL-4, 25 ng/mL IL-7, 25 ng/mL IL-9, 5 ug/mL IL-15, 100 ug/mL IL-21, 50 ug/mL IL-12, 0.25 mg/mL IL-18 or 100 U/mL IFN-αA.

Anti-TGF-β1 partially restores the surface expression of NKG2D and 2B4 on NK cells. Histograms correspond to NK cells from one representative donor treated with the isotype controls (green lines), medium alone (RPMI 1640 supplemented with 10% FBS in the presence of IL-15 (10 ng/ml) and IL-2 (100 U/ml)) (black lines) and TGF-β1 (1 ng/ml) (red lines).
Cells Peripheral blood lymphocytes were isolated from healthy female donors using Ficoll gradient. celltype'>celltype'>NK celltype'>cells were further purified by using the human celltype'>celltype'>NK cell isolation kit and the autoMACS instrument , according to the manufacturer's instructions. The identity of NK cells was confirmed by FACS. Purity was >98%. LILRB1-positive NK clones were grown in culture in the presence of 10 ng/ml IL-15 and then used for NK cytotoxicity assays.