Name: the neuroblast assay: an assay for the generation and enrichment of neuronal progenitor cells from differentiating neural stem cell progeny using flow cytometry
Uploaded: 2012-04-22T14:00:00
Description: Watch this Scientific Journal Video about The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytomet

Dette arbejde blev støttet med midler fra Overstreet Foundation.

1. Grundlæggende oprettes inden den går videre til Cell Culture <ol><li> NeuroCult NSC Basal Medium og NeuroCult NSC Proliferationsrespons Supplementer er blandet i et 9:1 forhold henholdsvis at komplet NSC medium. </li><li> En 37 ° C vandbad anvendes til at opvarme mediet. </li><li> Passende mængde af varme-inaktiveret føtalt kalveserum (FCS) optøs. </li><li> Passende mængde lager vækstfaktor opløsninger, herunder epidermal vækstfaktor (EGF ved 10 ug / ml), basisk fibroblast vækstfaktor (b-FGF ved 10 ug / ...

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	<li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M., Ansari, S., Reynolds, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457 (2011).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods. Mol. Biol. 750, 61-77 (2011).</li><li>Scheffler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+characterization+of+adult+brain+neuropoiesis.">Phenotypic and functional characterization of adult brain neuropoiesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 9353-9358 (2005).</li><li>Azari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+immature+neuronal+cells+from+neural+stem+cell+progeny.">Purification of immature neuronal cells from neural stem cell progeny.</a> PLoS One. 6, e20941-e20941 (2011).</li><li>Amariglio, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-Derived+Brain+Tumor+Following+Neural+Stem+Cell+Transplantation+in+an+Ataxia+Telangiectasia+Patient.">Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient.</a> Plos Medicine. 6, 221-231 (2009).</li></ol>

Evnen til at isolere defineret neurale cellepopulationer (dvs. neuroner, astrocytter og oligodendrocytter) kan løse nogle af de hindringer, der er forbundet med den kliniske anvendelse af neurale stamceller (NSC). For det første sætter os i stand til fuldt ud at karakterisere start befolkning donorceller. For det andet tillader os at anvende en bestemt celletype eller en kombination af forskellige celletyper med et specifikt forhold, afhængig af beskaffenheden eller stadiet af sygdommen. Endelig kan ved hjælp af h?...

<table border="1"><tr><td> Navn på reagenset </td><td> Type </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> NeuroCult NSC Basal Medium </td><td> Medium </td><td> Stem Cell Technologies </td><td> 05700 </td><td></td></tr><tr><td> NeuroCult NSC Proliferation Supplements </td><td> Mediesupplement </td><td> Stem Cell Technologies </td><td> 05701 </td><td></td></tr><tr><td> % 0,05 trypsin-EDTA </td><td> Reagens </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> Sojabønnetrypsininhibitor </td><td> Reagens </td><td> Sigma </td><td> T6522 </td><td></td></tr><tr><td> Pen / Strep </td><td> Reagens </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> * MEM </td><td> Reagens </td><td> Gibco </td><td> 41500-018 </td><td> HEM komponent </td></tr><tr><td> * HEPES </td><td> Reagens </td><td> Sigma </td><td> H4034 </td><td> HEM komponent </td></tr><tr><td> * Destilleret vand </td><td> Reagens </td><td> Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> Cellefilter </td><td> Sieve </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> T25 kolbe </td><td> Kultur ware </td><td> Nalge Nunc international </td><td> 136196 </td><td></td></tr><tr><td> T80 kolbe </td><td> Kultur ware </td><td> Nalge Nunc international </td><td> 178905 </td><td></td></tr><tr><td> 15 ml rør </td><td> Kultur ware </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> 50 ml rør </td><td> Kultur ware </td><td> BD Falcon </td><td> 352070 </td><td&gt; </td></tr><tr><td> EGF </td><td> Vækstfaktor </td><td> R &amp; D </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Vækstfaktor </td><td> R &amp; D </td><td> 3139-FB </td><td></td></tr><tr><td> Heparin </td><td> Vækstfaktor </td><td> Sigma </td><td> H4784 </td><td> Rekonstitueret i PBS </td></tr><tr><td> Anti-PSA-NCAM-PE </td><td> Antistof </td><td> Miltenyi Biotec </td><td> 130-093-274 </td><td></td></tr><tr><td> PE-konjugeret mus IgG1 </td><td> Isotypekontrolantistof </td><td> BD Pharminogen </td><td> 556029 </td><td></td></tr><tr><td> Heparin </td><td> Vækstfaktor </td><td> Sigma </td><td> H4784 </td><td> Rekonstitueret i PBS </td></tr><tr><td> HrBMP-4 </td><td> Vækstfaktor </td><td> R &amp; D </td><td> 314-BP-010 </td><td></td></tr><tr><td> Poly-L-OrnitHine </td><td> Reagens </td><td> Sigma </td><td> P4957 </td><td></td></tr><tr><td> Føtal kalveserum </td><td> Medium Supplement </td><td> Gibco </td><td> 10099-141 </td><td></td></tr><tr><td> GFAP </td><td> Antistof </td><td> DAKO </td><td> Z0334 </td><td></td></tr><tr><td> B-III tubulin </td><td> Antistof </td><td> Promega </td><td> G7121 </td><td></td></tr></table> * For at gøre HEM, mix 1 x 10 L pakke MEM og 160 ml 1M HEPES og bringe volumen til 8,75 L med destilleret vand. Indstilles det endelige pH til 7,4 og opbevares ved 4 ° C.

the neuroblast assay: an assay for the generation and enrichment of neuronal progenitor cells from differentiating neural stem cell progeny using flow cytometry

Neurale stamceller (NSC) kan isoleres og ekspanderes i stor skala ved hjælp af neurosfæren assayet og differentieret i de tre primære celletyper i centralnervesystemet (CNS), nemlig astrocytter, oligodendrocytter og neuroner. Disse egenskaber gør neurale stam-og progenitorceller en uvurderlig vedvarende kilde af celler til in vitro-undersøgelser, såsom lægemiddel-screening, neurotoxicology og elektrofysiologi og også for celle substitutionsterapi mange neurologiske sygdomme. I praksis er imidlertid heterogenitet NSC afkom, lav produktion af neuroner og oligodendrocytter, og overvægt af astrocytter efter differentiering begrænse deres kliniske anvendelser. Her beskriver vi en hidtil ukendt metode til generering og efterfølgende oprensning af umodne neuroner fra murine NSC afkom med fluorescensaktiveret cellesortering (FACS)-teknologi. Anvendelse af denne metode kan en stærkt beriget neuronal progenitorcellepopulation opnås vidHout nogen mærkbar astrocyt og bona fide NSC forurening. Fremgangsmåden omfatter differentiering af NSC afkom isoleres og ekspanderes fra E14 muse ganglioniske Eminencer anvendelse af neurosfæren assayet, efterfulgt af isolering og berigelse af umodne neuronale celler baseret på deres fysiske (størrelse og intern kompleksitet) og fluorescerende egenskaber ved hjælp af flowcytometri-teknologi. Alt i alt tager det 5-7 dage at generere neurosfærerne og 6-8 dage til at differentiere NSC afkom og isolere højtoprensede umodne nerveceller.

Watch this Scientific Journal Video about The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytomet

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Denne video protokol viser en hidtil ukendt fremgangsmåde til generering og efterfølgende oprensning af neuronale progenitorceller fra en fornyelig kilde af neurale stamceller (NSC) baseret på deres fysiske (størrelse og intern granularitet) og fluorescerende egenskaber ved hjælp af flowcytometri-teknologi.

Den neuroblast Assay: Et assay for generering og berigelse af neuronale progenitorceller fra Differentiering neural stamcelle Afkom anvendelse af flowcytometri

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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Neuroscience

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Isolering og dyrkning af neurale kamceller fra embryonale Murin Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Feeder-fri Udledning af neurale Crest stamceller fra menneskelige pluripotente stamceller

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flowcytometri Protokoller for Surface og intracellulære Antigen Analyser af neurale Cell Typer

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Celle Sortering af neurale stam- og progenitorceller fra voksne mus subventrikulære Zone og Live-billeddannelse af deres cellecyklus Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Enumeration of Neural Stem Cells Using Clonal Assays

Tælling af neurale stamceller Brug Klonale Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Identifikation af celleoverflade markører for primær neurale stamme og stamceller ved metabolisk mærkning af Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...