Optimized DNA sequence encoding Human SCF mature chain was expressed in Escherichia Coli.
Native human Stem Cell Factor is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately 18 kDa. Recombinant SCF is a monomer protein consisting of 165 amino acid residue subunits, and migrates as an approximately 18 kDa protein under non-reducing and reducing conditions in SDS-PAGE.
>97%, as determined by SDS-PAGE and HPLC
The ED(50) was determined by the dose-dependent stimulation of the proliferation of human TF-1 cells is ≤.0 ng/ml, corresponding to a specific activity of ≥ x units/mg.
MKKTQTWILT CIYLQLLLFN PLVKTEGICR NRVTNNVKDV TKLVANLPKD YMITLKYVPG MDVLPSHCWI SEMVVQLSDS LTDLLDKFSN ISEGLSNYSI IDKLVNIVDD LVECVKENSS KDLKKSFKSP EPRLFTPEEF FRIFNRSIDA FKDFVVASET SDCVVSSTLS PEKDSRVSVT KPFMLPPVA A SSLRNDSSSS NRKAKNPPGD SSLHWAAMAL PALFSLIIGF AFGALYWKKR QPSLTRAVEN IQINEEDNEI SMLQEKEREF QEV
Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Recombinant SCF was lyophilized from a.2 μm filtered solution in PBS pH7.0.
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.
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.
This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.
Cell transfection and plasmids
- Human immature thymocytes were obtained as previously described + cells followed by negative selection using the human CD4+ T-cell enrichment cocktail according to the manufacturer's instructions.
- The Tax expression plasmid, pCMV-TaxGFP (generous gift from R.…
Processing whole blood samples
- Peripheral (PB.1 and PB.2) and cord (CB.1 and CB.2) blood-derived CD34+ cells were obtained from AllCells .
- Blood collections were performed at AllCells and using standard, 8 ml Vacutainer Cell Processing Tubes (both sodium…
Erythroid precursor cultures
- After informed consent had been obtained, peripheral blood from CDA II patients and from 5 healthy control relatives was collected into sterile heparinised tubes.
- Light-density mononuclear cells obtained by centrifugation on density gradient were enriched for…
2.3. Differentiation of hESC
- H1 hESCs were plated at 3 × 106 per well of 6-well ultralow attachment (ULA) plates in a total volume of 4 mL of medium'>X-VIVO-15 medium , supplemented with 1 mM sodium pyruvate, nonessential…
Primary human cord blood-derived mast cells (CBMC) were generated from umbilical cord blood mononuclear cells using a modification of the method of Saito
et al.and characterized as previously described 6 cells/mL and passaged weekly in
- Frozen female CD34+ CBCs were supplied by Bio-Resource Center .
- C34+ CBCs were cultured in hematopoietic culture medium [serum-free X-Vivo10 containing 50 ng/mL IL-6 , 50 ng/mL sIL-6 , 50 ng/mL SCF , ten ng/mL…
- Cord blood-derived mast cells were derived in supplemented medium'>StemPro-34 SFM medium including 100 ng/mL recombinant human SCF (hSCF , , ) and 10 ng/mL human IL-6 as previously described
Gene Delivery into CD34+ Cells by Retrovirus
- The CD34+ cells isolated from cord blood were cultured in X-VIVO15 , supplemented with 1% human serum albumin (HSA) and stimulated with a cytokine cocktail [100 ng/ml stem cell factor, 100 ng/ml…
A human IgE preparation can induce IL-8 and IL-6 production and promote survival in human mast cells.
- Human CBMCs (1×105 cells) were incubated with 0 , 0.5, or 5 µg/mL of the indicated human IgEs in the absence of SCF and IL-6 for 72 hr.
- Fresh UCB was obtained from , and the use of the UCB samples was reviewed and approved by the institutional review boards (IRB) of each cord blood collection hospital as well as those of…