Mouse Interleukin-4 Recombinant

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Mouse Interleukin-4 Recombinant

$70.00$3,500.00


accession P07750


Source Optimized DNA sequence encoding Mouse Interleukin-4 mature chain was expressed in Escherichia Coli.
Molecular weight Native Mouse Interleukin-4, generated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calculated molecular mass of approximately 14 kDa. Recombinant Interleukin-4 is a monomer protein consisting of 121 amino acid residue subunits, migrates as an approximately 14 kDa 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 stimulation of the proliferation of human TF-1 cells is ≤.4 ng/ml, corresponding to a specific activity of ≥x units/mg.
Protein Sequence MGLNPQLVVI LLFFLECTRS HIHGCDKNHL REIIGILNEV TGEGTPCTEM DVPNVLTATK NTTESELVCR ASKVLRIFYL KHGKTPCLKK NSSVLMELQR LFRAFRCLDS SISCTMNESK STSLKDFLES LKSIMQMDYS
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 PBS, pH.
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 B-cell-activation
Molecular function Cytokine
Molecular function Growth-factor

Methods

In-vitro NK-DC co-culture

  • Whole blood samples were obtained from healthy laboratory donors for the isolation of purified and enriched NK and DC cell populations from PBMC using magnetic cell separation.
  • The development of an NK-DC co-culture system required two independent steps.
  • Firstly, DC were prepared using a pre-optimised magnetic cell sorting kit for the extraction of CD14+ monocytes from PBMC.
  • Following isolation, CD14+ monocytes were treated with 100 ng/ml of GM-CSF and 1000 U/ml of IL-4 in RPMI containing 5% autologous serum and maintained in a 5% CO2 cell culture incubator at 37°C for 5 days.
  • This altered their phenotype to that of immature dendritic cells (iDC) with a population of >90% CD14CD1a+ iDC cells, comparable with the published literature +CD16+ NK cell.
  • Purities of >98% were attained consistent with published literature 2 cell culture incubator at 37°C.
  • Co-culture cell ratios…
  • Whole blood samples were obtained from healthy laboratory donors for the isolation of purified and enriched NK and DC cell populations from PBMC using magnetic cell separation.
  • The development of an NK-DC co-culture system required two independent steps.
  • Firstly, DC were prepared using a pre-optimised magnetic cell sorting kit for the extraction of CD14+ monocytes from PBMC.
  • Following isolation, CD14+ monocytes were treated with 100 ng/ml of GM-CSF and 1000 U/ml of IL-4 in RPMI containing 5% autologous serum and maintained in a 5% CO2 cell culture incubator at 37°C for 5 days.
  • This altered their phenotype to that of immature dendritic cells (iDC) with a population of >90% CD14CD1a+ iDC cells, comparable with the published literature +CD16+ NK cell.
  • Purities of >98% were attained consistent with published literature 2 cell culture incubator at 37°C.
  • Co-culture cell ratios of 1∶1 and 1∶5 (NK∶DC) were then studied as these ratios are most favourable for DC maturation 6 of each cell type in a final media volume of 500 µl.
  • The control wells had either DC or NK cells in isolation.
  • Trans-well experiments were performed using the same conditions, but in the presence or absence of a 0.4 µm insert to separate NK cells and DC.
  • At the end of co-culture C were tested for the expression of the maturation markers C86 (, 2331 ) and HLA- and the chemokine receptor CC7 .
  • The DC gate on flow cytometry was defined by a combination of scatter plot and CD56 staining to identify NK cells.
  • The Δ Mean Fluorescence Intensity (Δ MFI) per experiment for CD86, HLA-DR and CCR7 was calculated as the difference in MFI for DC in co-culture with NK cells versus DC in isolation (background maturation).
  • Corresponding isotype controls were used.
  • Cells were analysed using a FACSCalibur flow cytometer with Winmdi 2.9 software , acquiring information from a total of 5,000 gated cells.

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Primary cell isolation and viral infection

  • Bone marrow macrophages (BMM) and bone marrow dendritic cells (BMDC) were generated as described previously

Immunoprotection analysis of Com1- and HspB-pulsed BMDCs

  • Mouse bone marrow dendritic cells (BMDCs, CD11c+) were isolated from the bone marrow of BALB/c mice, according to the protocol described previously [2.
  • Approximately 1 × 106 cells was added to each well of a six-well plate, and mouse GM-CSF (20 ng/ml) and IL-4 (10 ng/ml) was added to the culture medium every other day.
  • After 6 days of culture, BMDCs were stimulated with C.
  • burnetii I Ag (10 μg/ml), Com1 (10 μg/ml), HspB (10 μg/ml), E.
  • coli LPS (2 μg/ml), or 25 μl elution buffer (mock pulse) for 24 h at 37°C and 5% CO2.

mDCs inhibited Treg differentiation.

1 ng/ml IL-4
  • D–F, Bone marrow precursor cells freshly isolated from naïve mice were cultured in vitro in the presence of GM-CSF and IL-4.

T. crassiceps-inoculation induces elevation of IL-4 and hepatic expression of Ym-1.

specific IL-4
  • Six weeks post-inoculation, IL-4 and IFN-γ serum levels were determined from Tc-inoculated and non-inoculated mice by ELISA.

Antibodies and proteins

  • Antibodies directed against IκBα (#4814), phospho p38 (#9211), p38 (#9212), phospho ERK1/2 (#9106), ERK1/2 (#9102), phospho JNK1/2 (#9251), JNK1/2 (#9252) and phospho c-Jun (#3270) were all from Cell Signaling Technology.
  • Wnt5a antibody was from Biotechnology (Sc-30224), NFATC2 for Western blotting was from and for immunofluorescence from (Sc-13034), and the mouse monoclonal β-actin from .
  • LPS (from E.
  • Recombinant IL-4 and IL-13 proteins were bought from .
  • HlyA was obtained from Prof. S. Bhakdi (Mainz University, Germany).
  • HlyA was preincubated with polymyxin B (50 µg/ml) at 4°C for 30 min to remove any possible LPS contamination.

Functional assays

  • For cytokine signalling, LN T cells (5 × 106 cells/ml) were cultured 16 h with recombinant mouse IL-7 (10 ng/ml), IL-4 (40 ng/ml), IL-6 (45 ng/ml& ) or IL-15 (100 ng/ml& ) as previously described (

Immune modulation by IL-12 shRNA silenced DC.

recombinant mouse IL-4
  • Co-cultured T cells were harvested and gene expression of IFNγ, IL-2, IL-4 and IL-10 were detected by RT-PCR.

Spleen DC isolation and peripheral blood derived cultures

  • After experimentation the following protocol, adapted from mammalian methods 2 flasks with phenolic caps at a density of approximately 5×106 cells/ml of supplemented medium'>L-15 medium (20% FBS, 50 µg/mL gentamicin, and 0.25 µg/mL fungizone).
  • Cells were allowed to adhere for two hours, at which point non-adherent cells were washed off and new media was added containing mammalian IL-4 (250 U/ml, human:, ; :, , ) and GM-CSF (1000 U/ml, human:,; :).
  • Cells were “fed” additional IL-4 and GM-CSF (same concentration as above) on days two, four, and six.
  • Non-adherent cells removed at the 2 hr time point were transferred to a new flask for culture without addition of exogenous IL-4 or GM-CSF (or removal of non-adherent cells).
  • These mixed cultures (sans cytokines) yielded much the same result as the adherent culture with cytokines.
  • A more direct comparison of adherent cultures with or without exogenous cytokines was made.
  • No discernable…
  • After experimentation the following protocol, adapted from mammalian methods 2 flasks with phenolic caps at a density of approximately 5×106 cells/ml of supplemented medium'>L-15 medium (20% FBS, 50 µg/mL gentamicin, and 0.25 µg/mL fungizone).
  • Cells were allowed to adhere for two hours, at which point non-adherent cells were washed off and new media was added containing mammalian IL-4 (250 U/ml, human:, ; :, , ) and GM-CSF (1000 U/ml, human:,; :).
  • Cells were “fed” additional IL-4 and GM-CSF (same concentration as above) on days two, four, and six.
  • Non-adherent cells removed at the 2 hr time point were transferred to a new flask for culture without addition of exogenous IL-4 or GM-CSF (or removal of non-adherent cells).
  • These mixed cultures (sans cytokines) yielded much the same result as the adherent culture with cytokines.
  • A more direct comparison of adherent cultures with or without exogenous cytokines was made.
  • No discernable differences were seen (except perhaps small differences in the kinetics of the cultures), and addition of cytokines was subsequently abandoned.
  • Cells were cultured at RT in the atmosphere.

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Progressive acquisition of CD200R correlates with effector cytokine secretion.

IL-4
  • Intracellular cytokine staining with CD200R expression is shown for TNFα, IL-2, IL-4 and IFNγ.