Name: Human Interleukin-4 Recombinant (E.Coli)
SKU: RKP05112
Availability: InStock

Description

Methods (71)


accession	P05112

Source	Optimized DNA sequence encoding Human Interleukin-4 mature chain was expressed in E.Coli
Molecular weight	Native human Interleukin-4, generated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calculated molecular mass of approximately 15 kDa. Recombinant Interleukin-4 is a monomer protein consisting of 130 amino acid residue subunits, migrates as an approximately 15 kDa protein under non-reducingand reducing conditions in SDS-PAGE.
Purity	>95%, as determined by SDS-PAGE and HPLC
Biological Activity	The ED(50) was determined by the dose-dependent stimulation of the proliferation of human TF-1 cells is ≤.1 ng/ml, corresponding to a specific activity of >x units/mg.
Protein Sequence	MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS
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-4 was lyophilized from a.2 μm filtered solution in.5% glycine,.5% sucrose,.01% Tween80, mM Glutamic acid, pH.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	P24394 IL4RA_HUMAN
Biological Process	B-cell-activation
Molecular function	Cytokine
Molecular function	Growth-factor

Methods


Phenotype of clinical grade LAKs and DCs. DCs were generated by culture of CD14+ PBMCs with GMCSF and IL-4 for 5 days.
ICI 182,780 revert E2-inhibitory effect in the release of a-defensins 1-3 by DCs. CD14+ cells isolated from fresh blood were cultured for 5 days in the presence of IL-4 and GM-CSF to differentiate into DCs.
Isolation and culture of cells The adherence monocytes were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, , ), 50 ng/ml human IL-4 , and 50 ng/ml human granulocyte-macrophage colony-stimulating factor (GM-CSF) at 37°C and 5% CO₂. IL-4 and GM-CSF were added to the cultured cells every other day. After 5 days of culture, more than 95% of the cells had converted to immature HMDCs (iHMDCs), with a phenotype of CD11c⁺, CD54 ^low, CD83 ^low, CD40 ^low, CD58 ^low, CD86^low, and CD80^low, as determined by flow cytometry.
LMWHA inhibits IL-4 or IL-13-induced fibrocyte differentiation. Human PBMC were cultured in SFM, SFM with 1 ng/ml IL-4, SFM with 300 µg/ml LMWHA, or SFM with 1 ng/ml IL-4 and 300 µg/ml LMWHA, or SFM, SFM with 1 ng/ml IL-13, or SFM with 1 ng/ml IL-13 and 300 µg/ml LMWHA.
Generation of expanded DCs Conventional DCs were obtained from peripheral blood CD14⁺ cells as described previously, with minor modification. Briefly, CD14⁺ cells were cultured under GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in medium'>IMDM medium containing 10% FBS and 1% penicillin and streptomycin. CD14⁺ monocytes were obtained by negative selection. Cells were washed in PBS, and lineage antigen-positive (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, Glycophorin A) cells were removed by using the EasySep human monocyte enrichment kit without CD16 depletion . These lineage-negative CD14⁺ cells were cultured under 100 ng/ml human GM-CSF and 50 ng/ml human IL-4 in IMDM medium. Each cytokine was dissolved in PBS containing 0.1% BSA in 100 times the density used. For expansion, CD34⁺ cells were cultured under GM/SCF (100 ng/ml GM-CSF and 50 ng/ml SCF) or GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in IMDM medium. The medium was… Conventional DCs were obtained from peripheral blood CD14⁺ cells as described previously, with minor modification. Briefly, CD14⁺ cells were cultured under GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in medium'>IMDM medium containing 10% FBS and 1% penicillin and streptomycin. CD14⁺ monocytes were obtained by negative selection. Cells were washed in PBS, and lineage antigen-positive (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, Glycophorin A) cells were removed by using the EasySep human monocyte enrichment kit without CD16 depletion . These lineage-negative CD14⁺ cells were cultured under 100 ng/ml human GM-CSF and 50 ng/ml human IL-4 in IMDM medium. Each cytokine was dissolved in PBS containing 0.1% BSA in 100 times the density used. For expansion, CD34⁺ cells were cultured under GM/SCF (100 ng/ml GM-CSF and 50 ng/ml SCF) or GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in IMDM medium. The medium was exchanged every 3 or 4 days. Cells were cultured in the MPC treatment plate (MD6 with Lid Low-Cell Binding; Nalge Nunc, ). Read more
Generation of expanded DCs Conventional DCs were obtained from peripheral blood CD14⁺ cells as described previously, with minor modification. Briefly, CD14⁺ cells were cultured under GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in medium'>IMDM medium containing 10% FBS and 1% penicillin and streptomycin. CD14⁺ monocytes were obtained by negative selection. Cells were washed in PBS, and lineage antigen-positive (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, Glycophorin A) cells were removed by using the EasySep human monocyte enrichment kit without CD16 depletion . These lineage-negative CD14⁺ cells were cultured under 100 ng/ml human GM-CSF and 50 ng/ml human IL-4 in IMDM medium. Each cytokine was dissolved in PBS containing 0.1% BSA in 100 times the density used. For expansion, CD34⁺ cells were cultured under GM/SCF (100 ng/ml GM-CSF and 50 ng/ml SCF) or GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in IMDM medium. The medium was… Conventional DCs were obtained from peripheral blood CD14⁺ cells as described previously, with minor modification. Briefly, CD14⁺ cells were cultured under GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in medium'>IMDM medium containing 10% FBS and 1% penicillin and streptomycin. CD14⁺ monocytes were obtained by negative selection. Cells were washed in PBS, and lineage antigen-positive (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, Glycophorin A) cells were removed by using the EasySep human monocyte enrichment kit without CD16 depletion . These lineage-negative CD14⁺ cells were cultured under 100 ng/ml human GM-CSF and 50 ng/ml human IL-4 in IMDM medium. Each cytokine was dissolved in PBS containing 0.1% BSA in 100 times the density used. For expansion, CD34⁺ cells were cultured under GM/SCF (100 ng/ml GM-CSF and 50 ng/ml SCF) or GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in IMDM medium. The medium was exchanged every 3 or 4 days. Cells were cultured in the MPC treatment plate (MD6 with Lid Low-Cell Binding; Nalge Nunc, ). Read more
Comparison of the phenotype and IL-12p70 production of mature HOCl-oxidized lysate-loaded DCs generated in human AB serum-free or serum-containing AIM-V and CellGenix DC media, and supplementation with different concentrations of GM-CSF and IL-4. 500 IU/ml recombinant GM-CSF and 250 IU/ml recombinant IL-4 were sufficient concentrations for generating a favorable immature DC phenotype with low CD14, and intermediate HLA-DR and CD1c.
2.3. Differentiation of hESC H1 hESCs were plated at 3 × 10⁶ per well of 6-well ultralow attachment (ULA) plates in a total volume of 4 mL of medium'>X-VIVO-15 medium , supplemented with 1 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine (all PAA Laboratories GmbH) and 5 μM 2-mercaptoethanol . The following growth factors were added: 50 ng/mL recombinant human bone morphogenetic protein-4 (BMP-4, & ), 50 ng/mL recombinant human vascular endothelial growth factor , 20 ng/mL recombinant human stem cell factor , and 50 ng/mL recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF, & ). After 2-3 days, the medium was topped up with 2 mL of fresh supplemented X-VIVO-15 medium to produce a total volume of 6 mL. Subsequent feeding was performed every 2-3 days by replacing 2-3 mL of old medium with new supplemented X-VIVO-15 medium from which every 5 days a growth factor was removed starting with BMP-4 at day 5, followed by VEGF at… H1 hESCs were plated at 3 × 10⁶ per well of 6-well ultralow attachment (ULA) plates in a total volume of 4 mL of medium'>X-VIVO-15 medium , supplemented with 1 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine (all PAA Laboratories GmbH) and 5 μM 2-mercaptoethanol . The following growth factors were added: 50 ng/mL recombinant human bone morphogenetic protein-4 (BMP-4, & ), 50 ng/mL recombinant human vascular endothelial growth factor , 20 ng/mL recombinant human stem cell factor , and 50 ng/mL recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF, & ). After 2-3 days, the medium was topped up with 2 mL of fresh supplemented X-VIVO-15 medium to produce a total volume of 6 mL. Subsequent feeding was performed every 2-3 days by replacing 2-3 mL of old medium with new supplemented X-VIVO-15 medium from which every 5 days a growth factor was removed starting with BMP-4 at day 5, followed by VEGF at day 10 and SCF at day 15 of differentiation [ Read more
2.5. DC Maturation and Rapamycin Treatment Two days after monocytes were plated, monocyte-derived and hESC-derived immature DC were treated with 10 ng/mL and 5–7 ng/mL of rapamycin , respectively. On day 5, Cs were matured for 48 hr using a maturation cocktail consisting of 50 ng/mL of GM-CSF , 100 ng/mL IL-4 , 20 ng/mL IFNγ , 50 ng/mL TNFα , 10 ng/mL of IL-1β , and 1 μg/mL PGE₂. On day 6-7, DCs were harvested by gentle pipetting, passed through a 70 μm cell strainer, centrifuged, and resuspended prior to their use in experiments.
Generation of imDC In Vitro Monocytes and CD3⁺ T cells were isolated from PBMCs by using anti-CD14 and anti-CD3 microbeads respectively [⁺ cells and CD3⁺ T cells was consistently ranging from 93% to 97%, as determined by flow cytometry. Monocytes were cultured in RPMI supplemented with 10% human AB serum, 10 ng/ml IL-4, and 50 ng/ml GM-CSF for 7 days to generate imDC as we described before [

Human Interleukin-4 Recombinant (E.Coli)

Human Interleukin-4 Recombinant (E.Coli)

Description

Methods (71)

Methods

Phenotype of clinical grade LAKs and DCs.

ICI 182,780 revert E2-inhibitory effect in the release of a-defensins 1-3 by DCs.

Isolation and culture of cells

LMWHA inhibits IL-4 or IL-13-induced fibrocyte differentiation.

Generation of expanded DCs

Generation of expanded DCs

Comparison of the phenotype and IL-12p70 production of mature HOCl-oxidized lysate-loaded DCs generated in human AB serum-free or serum-containing AIM-V and CellGenix DC media, and supplementation with different concentrations of GM-CSF and IL-4.

2.3. Differentiation of hESC

2.5. DC Maturation and Rapamycin Treatment

Generation of imDC In Vitro

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