Rat Interleukin-4 Recombinant

HomeHematopoietins, IL-2 family, Recombinant Rat Cytokines

Rat Interleukin-4 Recombinant

$70.00 – $4,700.00

SKU: RKP20096 Categories: Hematopoietins, IL-2 family, Recombinant Rat Cytokines Tags: bsf-1, il-4, il4, interleukin-4, P20096

Description

Methods (6)


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.1 ng/µg(1EU/µg).
Presentation	Recombinant IL-4was lyophilized from a.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.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


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. Read more
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. Read more
Generation of bone marrow-derived DCs and PrA treatment 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… 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 supplemented with 20 ng/mL of lipopolysaccharide and 1 μg/mL of recombinant rat tumor necrosis factor-α . On day 9, cells were collected for phenotypic analysis by flow cytometric analyses and vaccination. Read more