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 arbeidet ble støttet av midler fra Overstreet Foundation.

1. Grunnleggende satt opp før du går videre til Cell Culture <ol><li> NeuroCult NSC Basal Medium og NeuroCult NSC spredning Kosttilskudd er blandet på en 9:01 ratio, henholdsvis, for å gjøre ferdig NSC medium. </li><li> En 37 ° C vannbad brukes til å varme opp medium. </li><li> Passende mengde varme inaktivert kalvefoster serum (FCS) er tint. </li><li> Passende mengde lager vekstfaktor løsninger inkludert epidermal vekstfaktor (EGF på 10 mikrogram / ml), grunnleggende fibroblastic vekstfaktor (b-FGF på 10 mi...

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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>

Muligheten til å isolere definerte nevrale celle populasjoner (dvs. nevroner, astrocytes og oligodendrocytes) kan løse noen av de hindringer knyttet til klinisk anvendelse av nevrale stamceller (NSCs). Først gjør det oss til å fullt ut karakterisere starter befolkning av donor cellene. For det andre gir det oss å bruke en bestemt celletype eller en kombinasjon av forskjellige celletyper med en bestemt ratio, avhengig av art eller stadium av sykdommen. Til slutt kan bruke høyanriket pre-differensierte nevrale cell...

<table border="1"><tr><td> Navn på reagensen </td><td> Type </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> NeuroCult NSC Basal Medium </td><td> Medium </td><td> Stilk Cell Technologies </td><td> 05700 </td><td></td></tr><tr><td> NeuroCult NSC spredning Supplements </td><td> Medium supplement </td><td> Stilk 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> Soybean trypsin inhibitor </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> * Er HEPES </td><td> Reagens </td><td> Sigma </td><td> H4034 </td><td> HEM komponent </td></tr><tr><td> * Destillert vann </td><td> Reagens </td><td> Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> Cell sil </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 internasjonal </td><td> 136196 </td><td></td></tr><tr><td> T80 kolbe </td><td> Kultur ware </td><td> Nalge Nunc internasjonal </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> Vekstfaktor </td><td> R &amp; D </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Vekstfaktor </td><td> R &amp; D </td><td> 3139-FB </td><td></td></tr><tr><td> Heparin </td><td> Vekstfaktor </td><td> Sigma </td><td> H4784 </td><td> Rekonstitueres i PBS </td></tr><tr><td> Anti-PSA-NCAM-PE </td><td> Antistoff </td><td> Miltenyi Biotec </td><td> 130-093-274 </td><td></td></tr><tr><td> PE-konjugert mus IgG1 </td><td> Isotype kontroll antistoff </td><td> BD Pharminogen </td><td> 556029 </td><td></td></tr><tr><td> Heparin </td><td> Vekstfaktor </td><td> Sigma </td><td> H4784 </td><td> Rekonstitueres i PBS </td></tr><tr><td> HrBMP-4 </td><td> Vekstfaktor </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> Kalvefoster Serum </td><td> Medium Supplement </td><td> Gibco </td><td> 10099-141 </td><td></td></tr><tr><td> GFAP </td><td> Antistoff </td><td> DAKO </td><td> Z0334 </td><td></td></tr><tr><td> B-III tubulin </td><td> Antistoff </td><td> Promega </td><td> G7121 </td><td></td></tr></table> * For å lage HEM, bland 1 x 10 L pakke MEM og 160 ml 1M HEPES og bringe volumet til 8,75 L med destillert vann. Sett den endelige PH til 7,4 og lagre den 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

Nevrale stamceller (NSCs) kan isoleres og utvidet i stor skala, med neurosphere analysen og differensiert i de tre store celletyper i sentralnervesystemet (CNS), nemlig astrocytes og oligodendrocytes og nevroner. Disse egenskapene gjør nevrale stilk og stamceller en uvurderlig fornybar kilde til celler for in vitro-studier som narkotika screening, neurotoxicology og elektrofysiologi og også for celle erstatning terapi i mange nevrologiske sykdommer. I praksis vil imidlertid heterogenitet av NSC avkom, lav produksjon av nevroner og oligodendrocytes, og overvekt av astrocytes følgende differensiering begrense sine kliniske anvendelser. Her beskriver vi en roman metode for generering og påfølgende rensing av umodne nerveceller fra murine NSC avkom med fluorescens aktivert celle sortering (FACS) teknologi. Ved hjelp av denne metoden, kan en høyanriket nevronale stamceller befolkning oppnås viddHout noen merkbar astrocyte og bona fide NSC forurensning. Prosedyren omfatter differensiering av NSC avkom isolert og utvidet fra E14 mus ganglionic eminenser bruker neurosphere analysen, etterfulgt av isolasjon og berikelse av umodne nevrale celler basert på deres fysiske (størrelse og intern kompleksitet) og fluoriserende egenskaper ved hjelp av flowcytometri teknologi. Samlet, tar det 5-7 dager å generere neurospheres og 6-8 dager til å differensiere NSC avkommet og isolere svært renset umodne nevronale celler.

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 videoen protokollen viser en ny metode for produksjon og etterfølgende rensing av nevrale stamceller fra en fornybar kilde til nevrale stamceller (NSCs) basert på deres fysiske (størrelse og intern granularitet) og fluorescerende egenskaper ved hjelp av flowcytometri teknologi.

Den Neuroblast analysen: en analyse for generering og berikelse av neuronal stamceller fra Skille Neural Stem Cell familie Bruke flowcytometrisystemer

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 ...

Research

JoVE Journal

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 kultur neural crest Celler fra Embryonic 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-free Utledning av Neural Crest stamceller fra menneskelige pluripotent 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

Flowcytometrisystemer protokoller for Surface og Intracellulær Antigen Analyser av Neural celletyper

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

Cell Sortering av Neural stilk og stamceller fra Adult Mouse subventricular Zone og Live-avbildning av deres Cell Cycle 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

Opplisting av Neural stamceller Bruke Klonale Analyser

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

Identifisere Cell Surface markører for primær neural Stem og stamceller ved metabolsk merking av 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 ...