Rat Interleukin-3 Recombinant

Rat Interleukin-3 Recombinant

$70.00$160.00


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 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 (

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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 CD11chiB220 and CD11clowB220+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: CD11chighB220DX5PDCA; pDCs: CD11clowB220+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.

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