Mouse Interleukin-4 Recombinant

HomeHematopoietins, Recombinant Mouse Cytokines

Mouse Interleukin-4 Recombinant

$70.00 – $3,500.00

SKU: RKP07750 Categories: Hematopoietins, Recombinant Mouse Cytokines Tags: bsf-1, il-4, il4, interleukin-4, P07750

Description

Methods (71)


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 0.2 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% CD14⁻CD1a⁺ 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% CD14⁻CD1a⁺ 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. Read more
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 × 10⁶cells 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. 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. 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. 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. Read more
Progressive acquisition of CD200R correlates with effector cytokine secretion. Intracellular cytokine staining with CD200R expression is shown for TNFα, IL-2, IL-4 and IFNγ.