Human Interleukin-6 Recombinant (E.coli)

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Human Interleukin-6 Recombinant (E.coli)

$70.00$2,700.00


accession P05231


Source Optimized DNA sequence encoding Human Interleukin-6 mature chain was expressed in E.Coli
Molecular weight Native human Interleukin-6 is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately kDa. Recombinant IL-6 is a homodimer protein consisting of amino acid residue subunits, and migrates as an approximately kDa protein under reducing conditions in SDS-PAGE.
Purity >97%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) was determined by the dose-dependent stimulation of the proliferation ofmurineTD1 cells was found to be less than.1 ng/ml, corresponding to specific activity ofx107 IU/mg.
Protein Sequence MNSFSTSAFG PVAFSLGLLL VLPAAFPAPV PPGEDSKDVA APHRQPLTSS ERIDKQIRYI LDGISALRKE TCNKSNMCES SKEALAENNL NLPKMAEKDG CFQSGFNEET CLVKIITGLL EFEVYLEYLQ NRFESSEEQA RAVQMSTKVL IQFLQKKAKN LDAITTPDPT TNASLLTKLQ AQNQWLQDMT THLILRSFKE FLQSSLRALR QM
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 Interleukin-6 was lyophilized from a.2 μm filtered PBS solution pH.0.
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.


Biological Process Acute-phase
Molecular function Cytokine
Molecular function Growth-factor

Methods

Cell Culture

  • Frozen female CD34+ CBCs were supplied by Bio-Resource Center .
  • C34+ CBCs were cultured in hematopoietic culture medium [serum-free X-Vivo10 containing 50 ng/mL IL-6 , 50 ng/mL sIL-6 , 50 ng/mL SCF , ten ng/mL TPO , and 20 ng/mL Flt3/4 ligand ].
  • Reprogrammed cells were cultured in feeder-less primate ES cell medium Repro FF (ReproCELL, cat.
  • No.
  • RCHEMD004), ReproFF2 (, .
  • RCHEMD006), mTeSR1 ( catalog number 05850) or E8 (16) supplemented with five ng/mL bFGF (total bFGF ten ng/mL) on Pronectin F-coated dishes.
  • Passage of human iPSCs was previously described

Cell cultures

  • Cord blood-derived mast cells were derived in supplemented StemPro-34 SFM medium including 100 ng/mL recombinant human SCF (hSCF , , ) and 10 ng/mL human IL-6 as previously described

Generation of DC

  • Blood samples from healthy donors are collected after informed consent, in accordance with the Declaration of Helsinki and approval of the Institutional Review Board of the University Hospital of the Ludwig-Maximilians-University, Munich, Germany.
  • Peripheral blood mononuclear cells (PBMC) are isolated by Ficoll density gradient centrifugation.
  • PBMC are resuspended in 15 ml VLE (very low endotoxin) RPMI 1640 medium supplemented with 1.5% human serum (DC medium) at 7.5′107 cells per 75 cm2 culture flask (NUNC, 178905) and incubated at 37°C and 5% CO2 for 1 h. Non-adherent cells are carefully removed by washing.
  • Adherent monocytes are cultured in medium containing 100 ng/ml GM-CSF (Leukine® by, ) and 20 ng/ml interleukin-4 ( 104-IL-050-CF) and fed with the same medium on days 3 and 6.
  • On day 6 of culture, the immature C are differentiated into mC by addition of medium containing 10 ng/ml IL-1β ( 201-LB-025-CF), 15 ng/ml IL-6 ( 206-IL-050-CF), 10 ng/ml TNFα (…
  • Blood samples from healthy donors are collected after informed consent, in accordance with the Declaration of Helsinki and approval of the Institutional Review Board of the University Hospital of the Ludwig-Maximilians-University, Munich, Germany.
  • Peripheral blood mononuclear cells (PBMC) are isolated by Ficoll density gradient centrifugation.
  • PBMC are resuspended in 15 ml VLE (very low endotoxin) RPMI 1640 medium supplemented with 1.5% human serum (DC medium) at 7.5′107 cells per 75 cm2 culture flask (NUNC, 178905) and incubated at 37°C and 5% CO2 for 1 h. Non-adherent cells are carefully removed by washing.
  • Adherent monocytes are cultured in medium containing 100 ng/ml GM-CSF (Leukine® by, ) and 20 ng/ml interleukin-4 ( 104-IL-050-CF) and fed with the same medium on days 3 and 6.
  • On day 6 of culture, the immature C are differentiated into mC by addition of medium containing 10 ng/ml IL-1β ( 201-LB-025-CF), 15 ng/ml IL-6 ( 206-IL-050-CF), 10 ng/ml TNFα ( 210-TA-050-CF) and 1 µg/ml PGE2 for 2 d.

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Purification and Transduction of Mouse and Human Hematopoietic Progenitors

  • Mouse Hematopoietic Progenitors (mLin) were purified from BM mononuclear cells of C57BL6/6J mice by .
  • The mouse cells were then prestimulated in StemSpam SFEM (serum-free expansion medium , ) with 50ng/mL mouse Stem Cell Factor (mSCF) , 100ng/mL human Interleukin-11 (hIL-11), 100ng/mL human Flt-3 Ligand (hFlt-3L) and 10ng/mL human Interleukin-3 (hIL-3) (ocky , , ) for 4-6 hours.
  • After which, mLin cells were transduced with the different lentivirus.
  • On the other hand, cord blood was collected from mothers attending the Royal Hospital, , after informed consent and via a protocol approved by the Local Research Ethics Committees.
  • Mononuclear cells (MNC) were obtained by Ficoll density centrifugation and ammonium chloride red cell lysis.
  • Density-separated CB MNCs were depleted for lineage marker positive cells via theSep™ system according to the manufacturer’s instructions to generate Lineage negative (Lin) cells.
  • Lin cells were pre-stimulated in and supplemented with 50ng/mL human Stem Cell Factor (hSCF), 50ng/mL human…
  • Mouse Hematopoietic Progenitors (mLin) were purified from BM mononuclear cells of C57BL6/6J mice by .
  • The mouse cells were then prestimulated in StemSpam SFEM (serum-free expansion medium , ) with 50ng/mL mouse Stem Cell Factor (mSCF) , 100ng/mL human Interleukin-11 (hIL-11), 100ng/mL human Flt-3 Ligand (hFlt-3L) and 10ng/mL human Interleukin-3 (hIL-3) (ocky , , ) for 4-6 hours.
  • After which, mLin cells were transduced with the different lentivirus.
  • On the other hand, cord blood was collected from mothers attending the Royal Hospital, , after informed consent and via a protocol approved by the Local Research Ethics Committees.
  • Mononuclear cells (MNC) were obtained by Ficoll density centrifugation and ammonium chloride red cell lysis.
  • Density-separated CB MNCs were depleted for lineage marker positive cells via theSep™ system according to the manufacturer’s instructions to generate Lineage negative (Lin) cells.
  • Lin cells were pre-stimulated in and supplemented with 50ng/mL human Stem Cell Factor (hSCF), 50ng/mL human Flt-3 Ligand (hFlt-3L), 20ng/mL human Thrombopoietin (hTPO) and 10ng/mL human Interleukin-6 (hIL-6) for 4-6 hours.
  • Lentiviral supernatants were added at a multiplicity of infection of 30 (for single transduction) or 20 (for each lentivirus during the double transduction).
  • All the transductions were carried out over-night in the presence of 4mg/mL of Polybrene .
  • The efficiency of transduction was analyzed at four days by eGFP or / and mCherry expression.

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Liquid Cultures assays

  • Two types of liquid culture were used one for maintaining stem/progenitors and the second for inducing myeloid differentiation.
  • For the maintenance of HSC/progenitors, transduced Lin cells were cultured in IMDM / 10% Fetal Calf Serum (FCS) supplemented with 20ng/mL SCF, 50ng/mL Interleukin-3 (IL-3), 20ng/mL IL-6 and 10ng/mL Granulocyte-Colony Stimulating Factor (G-CSF) .
  • Fresh media was added every week.
  • For Myeloid promoting differentiation, transduced cells were cultured as previously described for two weeks ( in IMDM/15% FCS supplemented with 2ng/ml IL-3 and 20ng/ml SCF.