Human Interleukin-6 Recombinant (E.coli)
Categories: HematopoietinsIL-6 gp130 familyRecombinant Human Cytokines$70.00 – $2,700.00
Description
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 21 kDa. Recombinant IL-6 is a homodimer protein consisting of 184 amino acid residue subunits, and migrates as an approximately 21 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 0.1 ng/ml, corresponding to specific activity of 1x107 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 0.1 ng/µg(1EU/µg).
Presentation
Interleukin-6 was lyophilized from a 0.2 μm filtered PBS solution pH7.0.
Reconstitution
A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than 0.1 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 at2° -8° 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
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Biological Process
Molecular function
Molecular function
Methods
Gene Delivery into CD34+ Cells by Retrovirus
- The CD34+ cells isolated from cord blood were cultured in X-VIVO15 , supplemented with 1% human serum albumin (HSA) and stimulated with a cytokine cocktail [100 ng/ml stem cell factor, 100 ng/ml Flt-3 ligand, 100 ng/ml thrombopoietin, and 100 ng/ml IL-6 ] in a 24-well plate (2×105 per well) for 48 h. The stimulated CD34+ cells were then harvested and placed into non-tissue culture-treated 6-well plates that had been coated with 20 µg/ml CH-296, a recombinant fibronectin fragment .
- (3×105 cells per well) in the presence of the respective virus supernatant.
- The virus supernatants were diluted 1∶2 with X-VIVO15 containing 1% HSA and the cytokine cocktail described above.
- Every 12 h, the medium was replaced with fresh virus supernatant.
- After 48 h of culture, the frequency of GFP- and/or Venus-expressing CD34+ cells was examined by FACS.
Clonogenic assay of primary tumor cells
- Mononuclear cells (MNCs) isolated from bone marrow samples using density gradient centrifugation were treated with IFN-γ and BCGV for 2 days.
- Then CD138+ and CD138− fractions were isolated from treated-MNCs using CD138 microbeads and an AutoMACS magnetic cell sorter .
- The CD138− fraction was further depleted of normal hematopoietic progenitors using CD34, CD3, CD4 and CD8 microbeads .
- The resulting two fractions (CD138+CD34−CD3−CD4−CD8− and CD138− CD34−CD3−CD4−CD8− cells; 0.5–2.5 × 105/ml) were plated with or without GV-Th2 cells at a ratio of 1:2–5 in a methylcellulose culture system , as described above for U266 cells, containing rhIL-6 (10 ng/ml).
- Tumor colonies were counted after 2–3 weeks of culture.
- The phenotype of the cells in these colonies was confirmed by flow cytometry.
Neural in vitro differentiation
-
Neural differentiation of LLC6P and LLC9P cells was performed as previously described
g for 5 minutes at 4°C and plated on polyornithine/laminin-coated cell culture dishes. - Passage number of LLC6P hpESCs at differentiation induction was 30–45; line LLC9P cells were used at passage numbers 48–60.
- Terminal differentiation of hpNSCs was performed in differentiation media containing DMEM/F12 (N2 supplement; 1∶50) and Neurobasal (B27 supplement; 1∶50) mixed at 1∶1 ratio.
- cAMP (300 ng/mL) was added to the media for 28 days.
- For induction of dopaminergic differentiation
CysLT1 receptor expression in human coronary artery smooth muscle cells is upregulated by pro-inflammatory stimuli.
- Real-time quantitative TaqMan RT-PCR for CysLT receptor mRNA in SMCs incubated in the absence and presence of LPS (10 μg/mL) for 1, 4, and 8 h (a) and IL-6 (20 ng/mL), TNF-α (10 ng/mL), or IFN-γ (20 ng/mL) for either 8 h (b) or 24 h (c).
DC Co-Cultures
- Sorted naïve (live, singlet, CD3+CD28+CD95-CCR7+CD27+) CD4+ T cells were co-cultured with sorted CD103+ DCs (live, lineage-CD14-HLA-DR+CD11c+) (100:1), sorted CD103− DCs (lineage-CD14-HLA-DR+CD11c+CD103-) (100:1), or anti-CD3/anti-CD28 beads (control, 4:1) in X-Vivo15 media under Th17 conditions (10 U/mL recombinant human IL-2, 12.5 ng/mL rhIL-1β, 25 ng/mL rhIL-21, 25 ng/ml rhIL-23, 10 μg/mL anti-IFNγ, 10 μg/mL anti-IL-12 and 2 ng/mL TGF-β, with or without 25 ng/mL rhIL-6).
- All cytokines were from , except IL-2, IL-6 and IL-21 .
- DC co-cultures were stimulated with SEB (1 μg/mL ).
- All cultures were fed on day 3 with rhIL-2 (2 U/mL), anti-IFNγ (10 μg/mL) and anti-IL-12 (10 μg/mL).
- After 7 days of co-culture, qRT-PCR was performed for
IL17A andRORc gene expression . - Gene expression was quantified using ΔΔCT analysis in excel (version 12.3.0).
Culture of human mast cells
- Human umbilical cord blood was collected from mothers who had normal uncomplicated deliveries at Tufts Medical Center.
- Human cord blood-derived cultured mast cells (hCBMCs) were prepared using hematopoetic stem cells (C34+) isolated by positive selection of C34+/AC133+ cells by magnetic cell sorting using an AC133+ cell isolation kit as previously reported [+ cells were grown in medium'>medium'>medium'>serum-free expansion medium'>medium , supplemented with 100 ng/ml recombinant human stem cell factor (rhSCF; kindly supplied by Orphan , , ), 100 U/ml penicillin, 100 μg/ml streptomycin and IL-3 for the first 3 weeks, then in the medium'>medium'>medium'>serum-free expansion medium'>medium with 50 ng/ml IL-6 (ocky , , ) and for 8 to 10 weeks, with fetal bovine serum (/Gibco, , , ) added from week 6.
- The purity of the hCBMCs was…
Baicalin up-regulates Foxp3 expression in HEK 293 T cells.
- HEK 293 T cells were treated with IL-6 in the presence or absence of Baicalin for 24 h, and IL-6R mRNA levels were examined by RT-PCR.
A human IgE preparation can induce IL-8 and IL-6 production and promote survival in human mast cells.
- IL-8, IL-6, and GAPDH mRNA levels were measured by quantitative RT-PCR.
Human Mast Cell Culture
- Human cord blood-derived cultured mast cells (hCBMCs) were cultured as described below.
- Human umbilical cord blood was collected following normal uncomplicated deliveries at Tufts Medical Center (Boston, MA) under Exemption #4 (discarded blood without identifiers) approved by the Tufts Medical Center Human Institutional Review Board (IRB).
- No consent form was required for discarded tissue.
- Cord blood cells were isolated by positive selection of C34+/AC133+ cells with magnetic cell sorting using an AC133+ cell isolation kit as previously reported + cells were suspended in StemSpan serum-free expansion medium , supplemented with 100 ng/ml rhSCF; kindly supplied by Sweden Orphan and 100 U/ml penicillin/100 µg/ml streptomycin , and IL-3 for the first 3 weeks, and 50 ng/ml IL-6 (ocky , ) for 8 to 10 weeks.
- Fetal bovine serum (FBS, , , ) was added from week 6 on.
- The purity of hCBMCs was evaluated…
Inflammatory conditions reduce Imatinib uptake and hMATE1 expression in hRASF.
- B, C, and D) Effect of 18 hours incubation with a TNFα, IL-1β and IL-6 (+sIL-6R) cocktail and with single cytokines (each at 10 ng/ml) on Imatinib uptake in hRASF , hMATE1-mRNA expression , hMATE1-protein expression by immunofluorescence staining (upper part of D) and by Western Blot analysis of biotynilated plasma membrane fractions hMATE1 (lower part of D showing an example of a typical Western blot together with the quantitative analysis of three independent experiments).
