Mouse FLT3 Ligand Recombinant

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Mouse FLT3 Ligand Recombinant

$70.00 – $3,500.00

SKU: RKP49772 Category: Recombinant Mouse Cytokines Tags: flt3 ligand, Flt3L, flt3lg, P49772, SL cytokine

Description

Methods (10)


accession	P49772

Source	Optimized DNA sequence encoding mouse FLT-3 ligand mature chain was expressed in Escherichia Coli.
Molecular weight	Native mouse FLT-3 Ligand, generated by the proteolytic removal of the signal peptide and propeptide, this molecule has a calculated molecular mass of approximately 19kDa. Recombinant FLT-3 Ligand is a monomer protein consisting of 163 amino acid residue subunits, and migrates as an approximately 19kDa protein under 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 proliferation of human AML5cells was found to be in the range of.0 ng/ml.
Protein Sequence	MTVLAPAWSP NSSLLLLLLL LSPCLRGTPD CYFSHSPISS NFKVKFRELT DHLLKDYPVT VAVNLQDEKH CKALWSLFLA QRWIEQLKTV AGSKMQTLLE DVNTEIHFVT SCTFQPLPEC LRFVQTNISH LLKDTCTQLL ALKPCIGKAC QNFSRCLEVQ CQPDSSTLLP PRSPIALEAT ELPEPRPRQ L LLLLLLLLPL TLVLLAAAWG LRWQRARRRG ELHPGVPLPS HP
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 FLT-3 Ligand was lyophilized from a.2 μm filtered solution withmM Tris,100mMNaCl, pH.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.

Molecular function	Cytokine

Methods


In vitro culture of Eos and MDSCs For obtaining Eos, naïve bone marrow cells were cultured in 10% medium'>FBS/PMI medium supplemented with stem cell factor and FLT3-ligand for 4 days, and then with medium containing recombinant mouse IL-5 . On day 12, the cells were collected for May-Grünwald-Giemsa staining and flow cytometry analysis⁶ cells per ml in 10% FBS/RPMI medium, supplemented with 40 ng ml⁻¹ GM-CSF. On day 4, the cells were collected for FACS analysis of CD11b⁺ CD11c⁻ Ly-6C⁺ Ly-6G^low MDSCs
Induction of 2nd iPSCs All isolated somatic cells were cultured in the presence of Dox (2 ug/ml) for induction of 2^nd iPSCs. Fibroblasts and hematopoietic cells were cultured in ES/iPSC medium. FLCD45 were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse EPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6, 10 ng/ml mouse Flt3 ligand, 10 ng/ml mouse GM-CSF, 10 ng/ml mouse VEGF and 50 ng/ml mouse SCF . HSCs, HPCs and MPs were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6 and 10 ng/ml mouse Flt3 ligand . Macrophages were cultured in the presence of 5 ng/ml M-CSF .
Induction of 2nd iPSCs All isolated somatic cells were cultured in the presence of Dox (2 ug/ml) for induction of 2^nd iPSCs. Fibroblasts and hematopoietic cells were cultured in ES/iPSC medium. FLCD45 were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse EPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6, 10 ng/ml mouse Flt3 ligand, 10 ng/ml mouse GM-CSF, 10 ng/ml mouse VEGF and 50 ng/ml mouse SCF . HSCs, HPCs and MPs were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6 and 10 ng/ml mouse Flt3 ligand . Macrophages were cultured in the presence of 5 ng/ml M-CSF .
Lentiviral transduction of bone marrow cells Freshly isolated bone marrow (BM) cells were plated in Iscove's Modified Dulbecco's Medium (IMDM) (31980, , ) containing 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, 200 mM glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 50 μM 2-mercaptoethanol, stem cell factor (SCF, 10 ng/ml, , ), Flt3-L (10 ng/ml), IL-11 (10 ng/ml), thrombopoietin (TPO, 10 ng/ml), IL-6 (10 ng/ml), and IL-3 (10 ng/ml). Bone marrow cells were spin-infected with 8 μg/ml polyprene and lentivirus (MOI of 0.1–0.5), at 2,500 rpm for 90 min. BM cells were then incubated for 3 hours at 37°C, counted and injected into NOD.SCID mice.
Eosinophil counting and culture Peripheral blood eosinophils were counted by’s staining [⁶/ml in IMDM containing 10% FBS , 100 IU/ml penicillin and 10 μg/ml streptomycin , 2 mM glutamine , and 50 μM 2-Mercaptoethanol supplemented with 100 ng/ml stem cell factor (SCF) and 100 ng/ml FLT3 ligand from days 0 to 4. On day 4, the medium was replaced with fresh medium containing 10 ng/ml recombinant mouse IL-5 (&systems). From this point forward, half of the medium was replaced every other day with fresh medium containing IL-5, and the cell density was maintained around 1×10⁶/ml. On day 14, the cells were harvested for in vitro experiments after flow cytometric identification by CC3-FITC and Siglec-F-PE (B ) staining as well as morphological examination by cytospun slide staining with a modified Giemsa preparation (iff ). As expected, more than 90% of harvested cells are eosinophils (data not shown).
Culture-derived Generation of Eosinophils from Bone Marrow Precursors Ex vivo generation by culture of mouse bone marrow-derived eosinophils was performed essentially as previously described by Dyer et al ^−/− and me^v mice by flushing the bone marrow cavity with RPM 1640 medium . After red cell lysis, the bone marrow cells were cultured at 10⁶/ml in medium containing RPMI 1640 with 20% fetal bovine serum (FBS, Cambrex, East Rutherford, ), 100 IU/ml penicillin, 10 µg/ml streptomycin, nonessential amino acids, 1 mM sodium pyruvate (all from , , ), and 50 µM 2-ME supplemented with 100 ng/ml stem cell factor (SCF) and 100 ng/ml FLT3 ligand (FLT3-L) from days 0 to 4. On day 4, the cells were moved to new flasks and the medium containing SCF and FLT3-L was replaced with medium containing 10 ng/ml recombinant mouse IL-5 . Every other day, from this point forward until cells were used, one-half of the… Ex vivo generation by culture of mouse bone marrow-derived eosinophils was performed essentially as previously described by Dyer et al ^−/− and me^v mice by flushing the bone marrow cavity with RPM 1640 medium . After red cell lysis, the bone marrow cells were cultured at 10⁶/ml in medium containing RPMI 1640 with 20% fetal bovine serum (FBS, Cambrex, East Rutherford, ), 100 IU/ml penicillin, 10 µg/ml streptomycin, nonessential amino acids, 1 mM sodium pyruvate (all from , , ), and 50 µM 2-ME supplemented with 100 ng/ml stem cell factor (SCF) and 100 ng/ml FLT3 ligand (FLT3-L) from days 0 to 4. On day 4, the cells were moved to new flasks and the medium containing SCF and FLT3-L was replaced with medium containing 10 ng/ml recombinant mouse IL-5 . Every other day, from this point forward until cells were used, one-half of the media was replaced with fresh media containing IL-5, and the concentration of the cells was adjusted each time to 10⁶/ml. Cells were enumerated in a hemocytometer and viability (consistently >90%) and purity were determined as mentioned above for human eosinophils. These methods yield eosinophils of normal morphology expressing proteins seen in mature eosinophils, including normal cell surface levels of Siglec-F Read more
AGM region explant culture E11.5 AGM regions were dissected and cultured for 5 days on floating 0.8 µm membranes at the liquid-gas interface with IMDM⁺ media consisting of 20% FCS, , IMDM and growth factors (100 ng/ml IL-3, 100 ng/ml SCF, and 100 ng/ml Flt3 ligand; all from) as previously described (
Lentiviral vectors, LSK-cell transduction, and differentiation and expansion of transduced macrophages Transduction, expansion, and differentiation of LSK cells into gene-corrected macrophages was done by adjusting the cytokine ‘cocktail’ mixture to optimize the culture conditions for each of four sequential stages, which included: 1) LSK transduction –murine SCF 50ng/ml, mIL-3 10 ng/ml, hFlt3-L 50 ng/ml and GM-CSF 10 ng/ml), culture time – two 12 hour periods; 2) progenitor expansion - mSCF 50 ng/ml, hFlt3-L 50 ng/ml and GM-CSF 10 ng/ml, culture time - 4 days; 3) macrophage lineage commitment - mSCF 1 ng/ml, hFlt3-L 1 ng/ml, GM-CSF 10 ng/ml, M-CSF 5 ng/ml, culture time - 3 days; and 4) macrophage differentiation - GM-CSF 10 ng/ml and M-CSF 5 ng/ml, culture - 4 days. StemSpan containing 2% FBS, 1% penicillin/streptomycin, 10 mM dNTP, and low density lipoprotein was used as the culture medium for the LSK transduction and DMEM with 10% FBS, 50 U/ml penicillin and… Transduction, expansion, and differentiation of LSK cells into gene-corrected macrophages was done by adjusting the cytokine ‘cocktail’ mixture to optimize the culture conditions for each of four sequential stages, which included: 1) LSK transduction –murine SCF 50ng/ml, mIL-3 10 ng/ml, hFlt3-L 50 ng/ml and GM-CSF 10 ng/ml), culture time – two 12 hour periods; 2) progenitor expansion - mSCF 50 ng/ml, hFlt3-L 50 ng/ml and GM-CSF 10 ng/ml, culture time - 4 days; 3) macrophage lineage commitment - mSCF 1 ng/ml, hFlt3-L 1 ng/ml, GM-CSF 10 ng/ml, M-CSF 5 ng/ml, culture time - 3 days; and 4) macrophage differentiation - GM-CSF 10 ng/ml and M-CSF 5 ng/ml, culture - 4 days. StemSpan containing 2% FBS, 1% penicillin/streptomycin, 10 mM dNTP, and low density lipoprotein was used as the culture medium for the LSK transduction and DMEM with 10% FBS, 50 U/ml penicillin and 50 ug/ml streptomycin was used for all other stages. Phenotype markers (F4/80, CD11b, CD11c) were analyzed by flow cytometry at each stage to monitor macrophage differentiation. Only adherent macrophages at the end of this procedure were used for PMT. Read more
Normal early thymocyte development in CBAP-deficient mice. DN thymocytes were isolated from 4-week-old littermates and co-cultured with OP9-DL4 cells in the presence of 1 ng/ml FLT3-ligand and 1 ng/ml IL-7.
Batf-3-dependent CD103+ DCs are major IL-12 producers. in the ear and 105 CD24hi cells from Flt3L BMDC cultures were transferred locally every 3 days starting at day 4 p.i.