Rat Interleukin-3 Recombinant

HomeHematopoietins, IL-3 family, CD markers, Recombinant Rat Cytokines

Rat Interleukin-3 Recombinant

$70.00 – $160.00

SKU: RKP04823 Categories: Hematopoietins, IL-3 family, CD markers, Recombinant Rat Cytokines Tags: il-3, IL3, interleukin-3, mcgf, P04823

Description

Methods (2)


accession	P04823

Source	Optimized DNA sequence encoding rat Interleukin-3 mature chain was expressed in Escherichia Coli.
Molecular weight	Native rat Interleukin-3 is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately kDa. Recombinant rat Interleukin-3 is a disulfide-linked monomer protein consisting of amino acid residue subunits, and migrates as an approximately kDa protein under non-reducing and 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 monkeyMBr-5 cells was found to be in the range of.0-40.0 ng/ml.
Protein Sequence	MVLASSTTSI LCMLLPLLML FHQGLQISDR GSDAHHLLRT LDCRTIALEI LVKLPVSGLN NSDDKANLRN STLRRVNLDE FLKSQEEFDS QDTTDIKSKL QKLKCCIPAA ASDSVLPGVY NKDLDDFKKK LRFYVIHLKD LQPVSVSRPP QPTSSSDNFR PMTVEC
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 rat Interleukin-3 was lyophilized from a.2 μm filtered PBS solution.
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.

Molecular function	Cytokine
Molecular function	Growth-factor

Methods


6-OHDA treatment and cytokine injection Animals were kept under pentobarbital sodium anesthesia (50 mg/kg) and placed in a stereotactic instrument . 6-OHDA was dissolved in saline containing ascorbic acid (10 μg/μL dissolved in 1% ascorbate-saline), kept on ice (4°C) and protected from light to minimize oxidation, until use. The rats were then given uni- or bilateral injections of 6-OHDA. Unilateral injection was employed for immunohistochemical analyses, and bilateral injection was used for all other studies. For unilateral injection, 5 μL of 6-OHDA was drawn into a Hamilton syringe and then injected into the right side of the striatum, through a hole made on the skull at 1 mm anterior to bregma and 3 mm lateral from the midline. The depth of the needle tip was 5 mm from the skull surface. The same amount of 6-OHDA was injected into the left side of the striatum for bilateral injection. The rate of fluid injection was 1 μL/min. The needle was left… Animals were kept under pentobarbital sodium anesthesia (50 mg/kg) and placed in a stereotactic instrument . 6-OHDA was dissolved in saline containing ascorbic acid (10 μg/μL dissolved in 1% ascorbate-saline), kept on ice (4°C) and protected from light to minimize oxidation, until use. The rats were then given uni- or bilateral injections of 6-OHDA. Unilateral injection was employed for immunohistochemical analyses, and bilateral injection was used for all other studies. For unilateral injection, 5 μL of 6-OHDA was drawn into a Hamilton syringe and then injected into the right side of the striatum, through a hole made on the skull at 1 mm anterior to bregma and 3 mm lateral from the midline. The depth of the needle tip was 5 mm from the skull surface. The same amount of 6-OHDA was injected into the left side of the striatum for bilateral injection. The rate of fluid injection was 1 μL/min. The needle was left at the point of injection for an additional 10 min after the injection and then slowly withdrawn. Because the bilaterally injected rats could not move well to drink or to eat, they were intraperitoneally injected with electrolyte solution (Solita-T3, Ajinomoto, Tokyo, Japan) twice per day for 1 week. A cytokine mixture containing 0.2 mg/mL rat recombinant GM-CSF and 0.2 mg/mL rat recombinant IL-3 was subcutaneously injected from the next day of the 6-OHDA-treatment at a dose of 10 μg/kg body weight ( Read more
DC Preparation and in vitro Challenge Enriched DCs were prepared by depleting T and B cells from total splenocytes using purified rat anti-mouse CD3 and anti-mouse CD19 mAbs, then anti-rat IgG magnetic beads , followed by further enrichment using CD11c⁺ magnetic beads (CD11c⁺ isolation kit ). The enriched CD11c⁺ DCs were seeded in a 96-well round-bottom tissue culture plate (5×10⁵ cells/well) in RPMI-1640 supplemented with 10% FCS, L-glutamine, Na pyruvate, Hepes, non-essential amino acids and 10⁻⁵ M β-ME with GM-CSF (10 ng/ml, ) and challenged with the TLR9 agonist, unmethylated CpG ODN 1668 (CpG; 6 µg/ml). The effect of injury on maturation of cDCs was examined at 24 h post-TLR9 activation and pDCs at 40 h post-activation. After activation, cells were washed and stained with APC-conjugated anti-mouse CD11c, PE Texas Red-conjugated anti-mouse B220, FITC-conjugated anti-mouse PDCA, PE-conjugated anti-mouse CD80, or CD86 and biotin-conjugated MHC Class-II with… Enriched DCs were prepared by depleting T and B cells from total splenocytes using purified rat anti-mouse CD3 and anti-mouse CD19 mAbs, then anti-rat IgG magnetic beads , followed by further enrichment using CD11c⁺ magnetic beads (CD11c⁺ isolation kit ). The enriched CD11c⁺ DCs were seeded in a 96-well round-bottom tissue culture plate (5×10⁵ cells/well) in RPMI-1640 supplemented with 10% FCS, L-glutamine, Na pyruvate, Hepes, non-essential amino acids and 10⁻⁵ M β-ME with GM-CSF (10 ng/ml, ) and challenged with the TLR9 agonist, unmethylated CpG ODN 1668 (CpG; 6 µg/ml). The effect of injury on maturation of cDCs was examined at 24 h post-TLR9 activation and pDCs at 40 h post-activation. After activation, cells were washed and stained with APC-conjugated anti-mouse CD11c, PE Texas Red-conjugated anti-mouse B220, FITC-conjugated anti-mouse PDCA, PE-conjugated anti-mouse CD80, or CD86 and biotin-conjugated MHC Class-II with streptavidin PE-Cy7 as secondary. cDCs and pDCs were analyzed as CD11c^hiB220⁻ and CD11c^lowB220⁺PDCA⁺, respectively, by flow cytometry. In some cases, cDCs and pDCs were further prepared from the enriched DC population to a higher purity of >98% using a BD FACSAria II cell sorter (cDCs: CD11c^highB220⁻DX5⁻PDCA⁻; pDCs: CD11c^lowB220⁺PDCA⁺). The sorted cDCs or pDCs were seeded in a 96-well U-bottom plate (2×10⁵ cells/well) and challenged with CpG (6 µg/ml). IL-3 (10 ng/ml) was added to the pDC culture to maintain viability. Supernatants were harvested 18–20 hr later and tested for levels of IL-6, IL-10, IFN-γ, TNF-α, and IL-12p70 using a Bead Array (CBA ) following the manufacturer’s instructions. IL-12p70 and IFN-α (PBL Interferon, , ) production was validated by ELISA assay. Read more