Human Interleukin-18 Recombinant

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Human Interleukin-18 Recombinant

$70.00$3,600.00


accession Q14116


Source Optimized DNA sequence encoding Human Interleukin-18 mature chain was expressed in Escherichia Coli
Molecular weight Native human Interleukin-18, generated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calculated molecular mass of approximately 18kDa. Recombinant Interleukin-18 is a monomer protein consisting of 157 amino acid residue subunits, migrates as an approximately 18 kDa protein under non-reducing and reducing conditions in SDS-PAGE.
Purity >92%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) was determined by the dose-dependent proliferation ofhuman PBMC co-stimulated with IL-12 cells was found to be in the range ofng/ml.

Protein Sequence YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKI
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 Interleukin-18 was lyophilized from.2 μm filtered PBS solution pH7.4 .
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 Q14116 IL18_HUMAN
Interactor Q9IW12
Interactor Q99665
Molecular function Cytokine

Methods

Regulation of CD69 and IFN-γ on NK cells.

IL-18
  • PBMCs from the three donors were incubated with 3D7 schizont-infected erythrocytes (iRBCs), with iRBCs plus human IFN-α or with a mixture of IL-12 and IL-18, or were leftuntreated in culture medium for 24 hours and analyzed by flow cytometry.

NK92/iRBCs co-culture and flow cytometry

  • NK92 cells were kept in two different environments for 24 hours prior to the co-culture: in normal cell medium'>medium medium'>(+rIL2; NK92 nm) and in cell medium'>medium without rIL-2 (starvation medium'>medium; NK92s).
  • Cells from both environments were co-cultured with 3D7 schizont-infected erythrocytes and uRBCs (NK92-RBCs ratio: 1:3) in their respective growth medium.
  • As a positive control, cells were also incubated with a mixture of IL-12 and IL-18 (and MBL, respectively; 100 ng/106 cells each).
  • After the indicated time of incubation at 37°C and 5% CO2, NK cells from the co-culture as well as cells incubated without RBCs were stained for 30 min at 4°C with fluorochrome-conjugated antibodies for surface CD56 (APC), CD3 (PE), CD16 (FITC), CD69 (FITC), CD25 (PE) in parallel with the appropriate isotype controls.
  • Cells were also internally stained with the IFN-γ (PE) antibody (all…
  • NK92 cells were kept in two different environments for 24 hours prior to the co-culture: in normal cell medium'>medium medium'>(+rIL2; NK92 nm) and in cell medium'>medium without rIL-2 (starvation medium'>medium; NK92s).
  • Cells from both environments were co-cultured with 3D7 schizont-infected erythrocytes and uRBCs (NK92-RBCs ratio: 1:3) in their respective growth medium.
  • As a positive control, cells were also incubated with a mixture of IL-12 and IL-18 (and MBL, respectively; 100 ng/106 cells each).
  • After the indicated time of incubation at 37°C and 5% CO2, NK cells from the co-culture as well as cells incubated without RBCs were stained for 30 min at 4°C with fluorochrome-conjugated antibodies for surface CD56 (APC), CD3 (PE), CD16 (FITC), CD69 (FITC), CD25 (PE) in parallel with the appropriate isotype controls.
  • Cells were also internally stained with the IFN-γ (PE) antibody (all ).
  • Dead cells were excluded from the analysis based on scatter signals and 7AAD fluorescence.
  • Acquisition of samples was carried out in a FACS canto flow cytometer .
  • Data were analysed with BD FACS Diva 6.0 software.
  • Gates were set on the events compatible to lymphocytes regarding "size of the cells" × "internal complexity" .
  • A total of 10.000 events were collected for each sample.

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Influence of selected cytokines and growth factors on the IL-33 synthesis in RA-SFs.

IL-18
  • RA-SFs (n=4) were stimulated for 24 h with TNF-α, IL-1β, IL-18, PDGF-BB or TGF-1β (concentrations as indicated in the figure).

Influence of selected cytokines and growth factors on the IL-33 synthesis in RA-SFs.

IL-18
  • RA-SFs (n=4) were stimulated for 24 h with TNF-α, IL-1β, IL-18, PDGF-BB or TGF-1β (concentrations as indicated in the figure).

Cell isolation and stimulation

  • For flow cytometry analysis and cell sorting of adult blood, fluorescently-labeled antibodies to the following antigens were used, CD3 (UCHT1), CD16 (3G8) CD56 (HCD56) and CD94 (DX22, , ); for cord blood, CD34 (581), CD117 (104D2), CD94 (HP-3D9), CD3 (UCHT1), and CD56 (NM16.2) .
  • medium'>NK cells (1 × 106) were incubated in 1 ml of medium'>NK complete medium for 24 h at 37°C in 5% CO2 with IL-2 (100 U/ml, Biologicalesources Branch, National Cancer Institute, Frederick, M), IL-12 (10 U/ml, , ) plus IL-18 (100 ng/ml& , , ), IL-15 (100 ng/ml), TGF-β (10 ng/ml& ), or PMA (10 ng/ml, ) plus the calcium ionophore, A23187 (250 ng/ml, , ).