Rat Interleukin-4 Recombinant

$70.00 – $4,700.00

SKU: RKP20096 Tags: bsf-1, il-4, il4, interleukin-4, P20096

Description

Accession

P20096

Source

Optimized DNA sequence encoding Rat Interleukin-4 mature chain was expressed in Escherichia Coli.

Molecular weight

Native Rat 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 126 amino acid residue subunits, migrates as an approximately 14 kDa protein under reducing conditions in SDS-PAGE.

Purity

>98%, 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

MGLSPHLAVT LFCFLICTGN GIHGCNDSPL REIINTLNQV TEKGTPCTEM FVPDVLTATR NTTENELICR ASRVLRKFYF PRDVPPCLKN KSGVLGELRK LCRGVSGLNS LRSCTVNEST LTTLKDFLES LKSILRGKYL QSCTSMS

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

Recombinant IL-4was lyophilized from a 0.2 μm filtered PBS solutionpH.5.

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.

Biological Process

B-cell-activation

Molecular function

Cytokine

Molecular function

Growth-factor

Methods

DC generation

Study was approved by the IRB of the Charles University, 2^ndMedical School.
Immature monocyte-derived DCs were generated as previously described [²culture flasks .
1 × 10⁷adherent monocytes were cultured for 5 days in culture media in the presence of GM-SF at the concentration of 500U/ml and 20 ng/ml ofL-4 [⁵D/ml and activated using Poly at 25 μg/ml, LPS at 1 μg/ml, or a cocktail of proinflammatory cytokines (L-1 andL-6, ).
Immature and mature DCs were used for further studies.

Adoptive transfer of cultured T cells

Spleens and lymph nodes (axillary and cervical) were collected from C57BL/6J female mice.
The spleens were mechanically disrupted, washed twice, and resuspended in cold PBS.
The lymph nodes were also mechanically disrupted and incubated on ice for 5 min followed by collection of the supernatant and centrifugation at 1400 r.p.m.
The pellet was then suspended in cold PBS.
These samples were filtrated with a 70-μm Cell Strainer and suspended in medium'>medium'>RPMI medium'>1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 μM sodium pyruvate, and 2.5 μM β-mercaptoethanol (medium'>medium'>RPMI growth medium).
CD4⁺ T cells were isolated by magnet sorting with anti-CD4 magnet beads according to the manufacturer's instructions .
CD4⁺ T cells were stimulated with anti-CD3ɛ/anti-CD28 antibodies coated on 24-well plates at a concentration of 5 μg/ml each.
Th1-conditioned cells were differentiated by the addition of…

Spleens and lymph nodes (axillary and cervical) were collected from C57BL/6J female mice.
The spleens were mechanically disrupted, washed twice, and resuspended in cold PBS.
The lymph nodes were also mechanically disrupted and incubated on ice for 5 min followed by collection of the supernatant and centrifugation at 1400 r.p.m.
The pellet was then suspended in cold PBS.
These samples were filtrated with a 70-μm Cell Strainer and suspended in medium'>medium'>RPMI medium'>1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 μM sodium pyruvate, and 2.5 μM β-mercaptoethanol (medium'>medium'>RPMI growth medium).
CD4⁺ T cells were isolated by magnet sorting with anti-CD4 magnet beads according to the manufacturer's instructions .
CD4⁺ T cells were stimulated with anti-CD3ɛ/anti-CD28 antibodies coated on 24-well plates at a concentration of 5 μg/ml each.
Th1-conditioned cells were differentiated by the addition of recombinant IL-2 (25 U/ml& , , , ), IL-12 (10 U/ml& ), and anti-IL-4 antibodies (25% culture supernatant of hybridoma; clone HB-188 Type , , , ).
Th2 polarization was initiated by the addition of recombinant IL-2 (25 U/ml&systems), IL-4 (100 U/mlocky , , ), and anti-IFN-γ antibodies (1% culture supernatant of hybridoma; clone HB-170Type Culture Collection).
Th17-conditioned cells were differentiated by the addition of recombinant IL-6 (20 ng/ml&systems), IL-23 (20 ng/ml&systems), TGF-β1 (3 ng/ml&systems), anti-IL-4 antibodies (25% culture supernatant of hybridoma; clone HB-188Type Culture Collection), and anti-IFN-γ antibodies (1% culture supernatant of hybridoma; clone CL-1975Type Culture Collection) in the presence of recombinant IL-2 (25 U/ml&systems) or anti-IL-2 antibody (10 μg/ml&systems).
These CD4⁺ T cells were diluted 1 : 3 for passage on day 2, and cultured with IL-2 and IL-12 for Th1; IL-2 and IL-4 for Th2; and IL-6, IL-23, TGF-β1 with or without IL-2 for Th17 in the same concentrations as above.
On day 4, these helper T cells were harvested and overlaid on Lympholyte M and centrifuged at 1000 × g for 20 min.
The intermediate layer was collected, washed with PBS, and resuspended in RPMI growth medium.
The cells were restimulated for 3 h with anti-CD3ɛ and anti-CD28 antibodies coated on 24-well plates.
After restimulation with anti-CD3ɛ and anti-CD28 antibodies, cells were harvested and washed twice with PBS.
Helper T cells (5.0 × 10⁶ or 1.0 × 10⁷) suspended in 500 μl PBS were injected i.p.
into mice at day 4 after SCI.
As a control, 500 μl PBS was injected into mice after SCI.
For IL-10 neutralization, Th1 cells were incubated with anti-IL-10 antibody (eBioscience; clone: JES5-16E3) or rat IgG (eBioscience, San Diego, CA, USA) at the concentration of 10 μg/ml, and were simultaneously restimulatd with anti-CD3ɛ and anti-CD28 antibodies.
After transfer of Th1 cells, 100 μg of anti-IL-10 antibody or rat IgG (eBioscience) was injected i.p.
into mice on SCI days 5, 7, and 11.

Spleens and lymph nodes (axillary and cervical) were collected from C57BL/6J female mice.
The spleens were mechanically disrupted, washed twice, and resuspended in cold PBS.
The lymph nodes were also mechanically disrupted and incubated on ice for 5 min followed by collection of the supernatant and centrifugation at 1400 r.p.m.
The pellet was then suspended in cold PBS.
These samples were filtrated with a 70-μm Cell Strainer and suspended in medium'>medium'>RPMI medium'>1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 μM sodium pyruvate, and 2.5 μM β-mercaptoethanol (medium'>medium'>RPMI growth medium).
CD4⁺ T cells were isolated by magnet sorting with anti-CD4 magnet beads according to the manufacturer's instructions .
CD4⁺ T cells were stimulated with anti-CD3ɛ/anti-CD28 antibodies coated on 24-well plates at a concentration of 5 μg/ml each.
Th1-conditioned cells were differentiated by the addition of…

Spleens and lymph nodes (axillary and cervical) were collected from C57BL/6J female mice.
The spleens were mechanically disrupted, washed twice, and resuspended in cold PBS.
The lymph nodes were also mechanically disrupted and incubated on ice for 5 min followed by collection of the supernatant and centrifugation at 1400 r.p.m.
The pellet was then suspended in cold PBS.
These samples were filtrated with a 70-μm Cell Strainer and suspended in medium'>medium'>RPMI medium'>1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 μM sodium pyruvate, and 2.5 μM β-mercaptoethanol (medium'>medium'>RPMI growth medium).
CD4⁺ T cells were isolated by magnet sorting with anti-CD4 magnet beads according to the manufacturer's instructions .
CD4⁺ T cells were stimulated with anti-CD3ɛ/anti-CD28 antibodies coated on 24-well plates at a concentration of 5 μg/ml each.
Th1-conditioned cells were differentiated by the addition of recombinant IL-2 (25 U/ml& , , , ), IL-12 (10 U/ml& ), and anti-IL-4 antibodies (25% culture supernatant of hybridoma; clone HB-188 Type , , , ).
Th2 polarization was initiated by the addition of recombinant IL-2 (25 U/ml&systems), IL-4 (100 U/mlocky , , ), and anti-IFN-γ antibodies (1% culture supernatant of hybridoma; clone HB-170Type Culture Collection).
Th17-conditioned cells were differentiated by the addition of recombinant IL-6 (20 ng/ml&systems), IL-23 (20 ng/ml&systems), TGF-β1 (3 ng/ml&systems), anti-IL-4 antibodies (25% culture supernatant of hybridoma; clone HB-188Type Culture Collection), and anti-IFN-γ antibodies (1% culture supernatant of hybridoma; clone CL-1975Type Culture Collection) in the presence of recombinant IL-2 (25 U/ml&systems) or anti-IL-2 antibody (10 μg/ml&systems).
These CD4⁺ T cells were diluted 1 : 3 for passage on day 2, and cultured with IL-2 and IL-12 for Th1; IL-2 and IL-4 for Th2; and IL-6, IL-23, TGF-β1 with or without IL-2 for Th17 in the same concentrations as above.
On day 4, these helper T cells were harvested and overlaid on Lympholyte M and centrifuged at 1000 × g for 20 min.
The intermediate layer was collected, washed with PBS, and resuspended in RPMI growth medium.
The cells were restimulated for 3 h with anti-CD3ɛ and anti-CD28 antibodies coated on 24-well plates.
After restimulation with anti-CD3ɛ and anti-CD28 antibodies, cells were harvested and washed twice with PBS.
Helper T cells (5.0 × 10⁶ or 1.0 × 10⁷) suspended in 500 μl PBS were injected i.p.
into mice at day 4 after SCI.
As a control, 500 μl PBS was injected into mice after SCI.
For IL-10 neutralization, Th1 cells were incubated with anti-IL-10 antibody (eBioscience; clone: JES5-16E3) or rat IgG (eBioscience, San Diego, CA, USA) at the concentration of 10 μg/ml, and were simultaneously restimulatd with anti-CD3ɛ and anti-CD28 antibodies.
After transfer of Th1 cells, 100 μg of anti-IL-10 antibody or rat IgG (eBioscience) was injected i.p.
into mice on SCI days 5, 7, and 11.

DCs were derived from the bone marrow precursors of Wistar rats as described previously ⁶ cells/ml in RPMI 1640 medium supplemented with 10 ng/ml each of rat GM-CSF and IL-4 .
To induce DC maturation, cells at day 6 in vitro were treated for 48 h with lipopolysaccharide (LPS, 10 ng/ml, , ).
The DC phenotype was confirmed by anti-OX62 labeling and flow cytometry using a FACS Calibur system (detailed below).
Only those preparations with a purity >85% DCs were used for subsequent experiments.
In other experiments, the DCs at day 6 were treated for 48 h with LPS plus different doses of PrA.

2.5. Primary splenocyte and bone marrow-derived dendritic cell (BMDC) culture

Immature BMDCs were isolated using a protocol modified from previous publications ⁶ cells) were then cultured in 1 ml complete medium for 7 d in the presence of recombinant rat IL-4 (10 ng/ml, , , ) and recombinant rat granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng/ml, , , ).
At day 7, immature BMDCs were resuspended in complete medium for maturation.
Mature BMDCs (1×10⁶ cells/well) were incubated with LPS (1 µg/ml, from E.
coli, , , ) for 12 h and then treated with culture supernatant of F.
prausnitzii (1 µl), sodium butyrate solution (0.005834 µmol) and denatured F.
prausnitzii or longum bacteria (1×10⁶ CFU/well, BMDCs: bacteria = 1:1) at 37°C for another 12 h. PBS treated BMDCs were used as a control.
Each group treatment was repeated four times.
The supernatant was collected and stored at −20°C.

Culture and characterization of DCs

DCs were generated from rat bone marrow as previously described[⁶ cells/mL in medium'>RPMI-1640 medium containing 10% fetal bovine serum , 2 mM glutamine, 100 μg/mL penicillin , 100 μg/mL streptomycin , 500 U/mL of recombinant rat granulocyte-macrophage colony- stimulating factor , and 500 U/mL of recombinant rat interleukin-4 .
On days 2 and 4, half of the culture supernatant was removed and replaced with fresh medium containing recombinant rat granulocyte macrophage colony- stimulating factor and interleukin-4.
On day 6, all the loosely adherent cells were harvested, centrifuged and counted.
Then C6 glioma cell lysates and DCs (a ratio of 3 tumor cells to 1 DC) were incubated in fresh medium with recombinant rat granulocyte macrophage colony-stimulating factor and interleukin-4 together at 37°C in 5% CO₂.
To stimulate DC maturation in vitro, on day 8, the cultures were additionally…

Rat Interleukin-4 Recombinant

Description

Methods

DC generation

Adoptive transfer of cultured T cells

Adoptive transfer of cultured T cells

Generation of bone marrow-derived DCs and PrA treatment

2.5. Primary splenocyte and bone marrow-derived dendritic cell (BMDC) culture

Culture and characterization of DCs

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Description

Methods

DC generation

Adoptive transfer of cultured T cells

Adoptive transfer of cultured T cells

Generation of bone marrow-derived DCs and PrA treatment

2.5. Primary splenocyte and bone marrow-derived dendritic cell (BMDC) culture

Culture and characterization of DCs

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