Human Ciliary Neurotrophic Factor Recombinant

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Human Ciliary Neurotrophic Factor Recombinant

$70.00 – $2,700.00

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SKU: RKP26441 Categories: , , , Tags: ,

Description

Methods (2)
accession P26441
Source Optimized DNA sequence encoding Human Ciliary Neurotrophic Factor mature chain was expressed in Escherichia Coli.
Molecular weight Native human CNTF has a calculated molecular mass of approximately22 kDa. Recombinant CNTF is a monomer protein consisting of 200 amino acid residue subunits, and migrates as an approximately 22kDa protein under non-reducing and 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 proliferation of Human TF1cells was found to be in the range of ng/ml.
Protein Sequence MAFTEHSPLT PHRRDLCSRS IWLARKIRSD LTALTESYVK HQGLNKNINL DSADGMPVAS TDQWSELTEA ERLQENLQAY RTFHVLLARL LEDQQVHFTP TEGDFHQAIH TLLLQVAAFA YQIEELMILL EYKIPRNEAD GMPINVGDGG LFEKKLWGLK VLQELSQWTV RSIHDLRFIS SHQTGIPARG SHYIANNKKM
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 Ciliary Neurotrophic Factor was lyophilized from a.2 μm filtered PBS 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.
Interactor P26992
Interactor P42702 LIFR_HUMAN
Biological Process Differentiation
Biological Process Neurogenesis
Molecular function Developmental-protein
Molecular function Growth-factor

Methods

Culture and In Vitro Differentiation of Human iPSCs

  • For in vitro differentiation, iPSC colonies were detached from the feeder layers en bloc using a dissociation solution (0.25% trypsin, 100 μg/ml collagenase IV [], 1 mM CaCl2, and 20% KSR; day 0) and cultured in suspension in…
  • For in vitro differentiation, iPSC colonies were detached from the feeder layers en bloc using a dissociation solution (0.25% trypsin, 100 μg/ml collagenase IV [], 1 mM CaCl2, and 20% KSR; day 0) and cultured in suspension in bacteriological dishes to form EBs in a humidified atmosphere of 3% CO2.
  • From day 1 to 4 of EB formation, 3 μM dorsomorphin , 3 μM SB431542 , and 3 μM BIO ((2′Z, 3′E)-6-bromoindirubin-3′-oxime) were added.
  • In addition, 1 μM retinoic acid and 1 μM purmorphamine were added on days 4 and 7, respectively, and maintained thereafter until day 16 .
  • The medium was changed every 2 days.
  • On day 16, the EBs were enzymatically dissociated into single cells using TrypLE Select , and the dissociated cells were cultured in suspension at a density of 1 × 105 cells/ml in proliferation medium consisting of medium'>serum-free medium (media hormone mix [MHM]; 2.
  • The medium was changed every 4∼6 days for approximately 15∼20 days to form the first neurospheres.
  • To passage neurospheres, the first neurospheres were dissociated in the same manner as described above and cultured at a density of 1 × 105 cells/ml in proliferation medium without purmorphamine for approximately 15∼20 days.
  • To assay neurosphere differentiation, undissociated 5–7 neurospheres were plated onto coverslips 10 mm in diameter coated with poly-L-ornithine and growth-factor-reduced Matrigel (50× dilution, thin coated), and cultured in differentiation medium that consisted of MHM supplemented with 2% B27 supplement, NEAA, 60 ng/ml T3, 10 ng/ml hLIF , and 25 ng/ml CNTF for 2–6 weeks in a humidified atmosphere of 5% CO2.
  • Half of the medium was changed every 2 or 3 days.
  • For the quantitative analysis of the differentiation efficiency into OL lineage cells, the numbers of neurosphere colonies containing more than 40 marker-positive cells (≥40 cells, oligodendrocyte [++]), those containing less than 40 marker-positive cells (1–39 cells, oligodendrocyte [+]), and those without marker-positive cells (oligodendrocyte [−]) were counted and are presented as the percentage of total neurosphere colonies.
  • To examine the expression of ER-stress markers, O4+ differentiated cells were purified 4 weeks after the attachment of the neurospheres using MACS technology with an anti-O4 antibody.

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Astrocyte culturing and differentiation

  • Astrocytes were differentiated from iPSCs as described.
  • Briefly, NPCs were cultured in MEM/F12 supplemented with 1% N2 , 0.1% B27 , 1% nonessential amino acids , 1% penicillin/streptomycin , 1% GlutaMAX solution…
  • Astrocytes were differentiated from iPSCs as described.
  • Briefly, NPCs were cultured in MEM/F12 supplemented with 1% N2 , 0.1% B27 , 1% nonessential amino acids , 1% penicillin/streptomycin , 1% GlutaMAX solution , 20 ng/mL LIF and 20 ng/mL EGF for 4–6 weeks.
  • Spheres were then transferred to MEM/F12 supplemented with 1% N2, 0.1, % B27, 1% nonessential amino acids, 1% penicillin/streptomycin, 1% GlutaMAX, 20 ng/mL FGF-2 and 20 ng/mL EGF , and mechanically passaged every 2 weeks.
  • To generate monolayer cultures, spheres were dissociated with papain and plated in wells coated with Matrigel ( 1:80).
  • Monolayers were split with Accutase approximately every 3–4 d, or when confluent.
  • ifferentiated astrocytes were created by culturing the cells in medium'>Neurobasal medium supplemented with 0.2% B27, 1% nonessential amino acids, 1% penicillin/streptomycin, 1% GlutaMAX solution, and 10 ng/mL CNTF for 2 weeks.
  • Before survival analysis, astrocytes were dissociated with Accutase and cultured at a density of 2×104 cells per well of a 96-well plate.
  • 2–3 days later, differentiated astrocyte cultures were transfected with pGW1-mApple using Lipofectamine 2000, as described above.

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