Rat Ciliary Neurotrophic Factor Recombinant

///Rat Ciliary Neurotrophic Factor Recombinant

Rat Ciliary Neurotrophic Factor Recombinant

$70.00$2,700.00

SKU: RKP20294 Category: Tags: ,

accession P20294


Source Optimized DNA sequence encoding Human Ciliary Neurotrophic Factormature chain was expressed in Escherichia Coli.
Molecular weight Native humanCNTF has a calculated molecular mass of approximately22 kDa. Recombinant CNTF is a monomer protein consisting of200 amino acid residue subunits, and migrates as an approximately kDa 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 ofHuman 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 Rat 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.


Biological Process Differentiation
Biological Process Neurogenesis
Molecular function Developmental-protein
Molecular function Growth-factor

Methods

Preparation of Primary Hippocampal Cultures

  • Primary hippocampal cultures were prepared from wild-type neonatal (E19) rat embryos (timed pregnant Sprague Dawley rats were obtained from Charles River Laboratories, Wilmington, MA) as described previously

RGC survival under normoxia and hypoxia Survival of RGC under normoxia and hypoxia (0.2% O2).

CNTF
  • RGC survival (ratio of living cell vs. total cells) was measured after treatment with 0, 1, 5 and 25 ng/ml PEDF or 10 ng/ml CNTF (n = 3; treated versus normoxic or hypoxic PEDF-free cultures, *P < 0.05, **P < 0.01; control hypoxic versus normoxic PEDF-free control culture, **°°P < 0.01).

Immunoisolation and Culture of RGC from Postnatal Mouse Retinae

  • Seven days old mice were killed according to institutional guidelines.
  • To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
  • The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
  • Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
  • Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
  • Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
  • Positive cell isolation was then achieved by incubating the filtrated…
  • Seven days old mice were killed according to institutional guidelines.
  • To isolate RGC, retinae were dissected and incubated for 45 min at 37°C in Dulbecco’s phosphate buffered saline (D-PBS; containing 160 U/ml papain, 200 U/ml DNAse.
  • The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor , 650 U/ml DNAse and 1:75 rabbit anti-rat macrophage antibody.
  • Cells were spun down (800′g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V).
  • Cell suspension was filtered through a nylon-mesh ( 20 , , , ) and sequentially added to immunopanning plates.
  • Briefly, immunopanning was performed using two subtraction plates (150-mm diameter petri dishes , , ) coated with goat anti-rabbit-IgG ( , , ; 10 μg/ml) to remove microglial cells after incubation of the supernatant with anti-macrophage antibody (WAK Chemie, ).
  • Positive cell isolation was then achieved by incubating the filtrated supernatant for 45 min on a 100-mm diameter petri dish sequentially pre-coated with goat anti-mouse-IgG and anti-Thy1.2 (mouse IgM, clone F7D5, , ).
  • Non-adherent cells were thoroughly washed off, and the bound cells were released by trypsination (12,000 U/ml in Hank’s buffered salt solution [HBSS] for 10 min in 5% CO2 at 37°C) and resuspended in culture medium.
  • The average yield, in thousands, of RGC per animal was 27.8 (±3.3), n = 11.
  • RGC were plated at 600 cells mm−2 on glass coverslips (10 mm in diameter) centered on 12-well tissue culture plates , which were pre-coated with 5 μg/ml poly-d-lysine (MW–40 kDa).
  • Neurons were cultured for a week at 37°C, 5% CO2, 95% humidity in medium'>Neurobasal medium supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) and pyruvate (1 mM), glutamine (2 mM), N-acetyl-l-cysteine (60 μg/ml), putrescine (16 μg/ml), selenite (40 ng/ml), bovine serum albumin (100 μg/ml; fraction V, crystalline grade), triiodothyronine (40 ng/ml), holotransferrin (100 μg/ml), dibutyryl cyclic AMP (250 μM), insulin (5 μg/ml), progesterone (62 ng/ml), B27 (1:50), d-mannose (50 μM), brain-derived neurotrophic factor (BDNF; 25 ng/ml, , ), ciliary neurotrophic factor (CNTF; 10 ng/ml) and forskolin (10 μM).
  • Cell viability was checked by examining the cell cultures under a phase contrast microscope (AxioVert25, Carl Zeiss, Jena, Germany) and determining cell adherence to the cover slip and the fraction of cells developing neurites.
  • We waited a week in order to let RGC develop a massive neuritic arborisation.
  • Since we were interested in the effect of PEDF on neuronal survival rather than on neurite growth, only preparations exhibiting an important development of neurites were selected for further tests.

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Knockdown of AMIGO3 promote RGC neurite outgrowth in the presence of inhibitory concentration of CME.

CNTF
  • Representative photomicrographs of βIII-tubulin+ RGC neurite outgrowth in CNTF and siRNA transfected retinal cells in the presence and absence of CME with or without CNTF.