Name: isolation and culture of rat embryonic neural cells: a quick protocol
Uploaded: 2012-05-24T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Vi takker Jonna Ellis for redaksjonell hjelp. Prosjektet beskrives ble støttet av Award Antall R01MH079751 (PI: F. Peruzzi) fra National Institute of Mental Health. Innholdet er utelukkende ansvaret til forfatterne og ikke nødvendigvis representerer den offisielle syn av National Institute of Mental Health eller National Institutes of Health.

1. Poly-D-Lysine (PDL): Forberedelse <ol><li> Tilsett 5 ml sterilt DDH 2 O til 5 mg PDL å oppnå en stamløsning av 1 mg / ml. </li><li> Bland stamløsning av pipettere flere ganger. </li><li> Brukes umiddelbart eller lagre Poly-D-Lysine Solution ved 2-8 ° C. </li></ol> 2. Poly-D-Lysine (PDL): Coating Plast Cell Culture Dishes <ol><li> Fortynn PDL stamløsning med steril DDH 2 O til endelig konsentrasjon på 10 mikrogram / ​​ml. </li><li> Pipetter nok løsning i et...

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Metoden for disseksjon og kultur av rotter hippocampus og kortikale nevroner beskrevet her kan utføre eksperimenter med nesten rene nevrale kulturer dyrket i en kjemisk definert medium (figur 3). Selv protokoller for dyrking nesten rene nevroner i serum-frie medier har tidligere blitt beskrevet 2,3,4, det er viktige endringer i vår metode. Forskjellig fra tradisjonelle protokoller (dvs. Banker et al.) 5, vi har erstattet trypsin med TrypLE Express, en mer skånsom dissosiasjon en...

<table border="1"><tbody><tr><td> Reagens </td><td> Konsentrasjon </td></tr><tr><td> Neurobasal </td><td> 98% </td></tr><tr><td> B27 </td><td> 2% </td></tr><tr><td> Glutamax </td><td> 0,5 mm </td></tr></tbody></table> Tabell I. Neurobasal/B27 komplett medium. <table border="1"><tbody><tr><td> Reagens </td><td> Konsentrasjon </td></tr><tr><td> Glukose </td><td> 16 mm </td></tr><tr><td> Sukrose </td><td> 22 mm </td></tr><tr><td> HEPES </td><td> 10 mM </td></tr><tr><td> NaCl </td><td> 160 mm </td></tr><tr><td> KCl </td><td> 5 mm </td></tr><tr><td> Na 2 HPO 4 </td><td> 1 mm </td></tr><tr><td> KH 2 PO 2 </td><td> 0,22 mM </td></tr><tr><td> Gentamicin </td><td> 50 mikrogram / ml </td></tr&gt; <tr><td> Fungizone </td><td> 250 ng / ml </td></tr><tr><td> pH </td><td> 7.4 </td></tr><tr><td> Osmolaritet </td><td> 320-330 mOsm </td></tr></tbody></table> Tabell II. Disseksjon medium. <table border="1"><tbody><tr><td> Reagens </td><td> Volum (mL) </td></tr><tr><td> Neurobasal/B27 komplett medium </td><td> 240 </td></tr><tr><td> Trypan blåved 0,4% </td><td> 250 </td></tr><tr><td> Total </td><td> 490 </td></tr></tbody></table> Tabell III. 50x Telle løsning. <table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Cat. nummer </td></tr><tr><td> Hibernate E </td><td> Brainbits </td><td> 767171 </td></tr><tr><td> Neurobasal </td><td> Gibco, Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 </td><td> Gibco, Invitrogen </td><td> 17504-044 </td></tr><tr><td> Fungizone </td><td> Gibco, Invitrogen </td><td> 15290-018 </td></tr><tr><td> Gentamicin sulfate </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> Glutamax 200 mm </td><td> Gibco, Invitrogen </td><td> 35050 </td></tr><tr><td> TrypLE Express w / o fenol rødt </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Cytosin-β-D-arabinofuranoside hydrochloride </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poly-D-Lysine </td><td> Sigma Aldrich </td><td> P6407 </td></tr><tr><td> Laminin 1 mg / ml </td><td> Millipore </td><td> CC095 </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3375 </td></tr><tr><td> Trypan blåved 0,4% </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tabell IV. Spesifikke reagenser. <table border="1"><tbody><tr><td> Utstyr </td><td> Firma </td><td> Cat. nummer </td></tr><tr><td> Stereo Microscope </td><td> Olympus </td><td> SZ61 </td></tr><tr><td> Store tang </td><td> FST </td><td> 11022-14 </td></tr><tr><td> Tynn tang </td><td> Moria </td><td> MC40B </td></tr><tr><td> Mikro tynn tang </td><td> Moria </td><td> MC31 </td></tr><tr><td> Knivskarpe saks </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Micro dissekere saks </td><td> FST </td><td> 91460-11 </td></tr><tr><td> Micro Dissecting Buede saks </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Glass 2-kammer lysbilder </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> 60 mm retter </td&gt; <td> BD Falcon </td><td> 353002 </td></tr><tr><td> 100 mm retter </td><td> Corning </td><td> 430167 </td></tr><tr><td> 15 ml rør </td><td> BD Falcon </td><td> 352099 </td></tr><tr><td> 1,5 ml Cryo-tube hetteglass </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Tabell V. Utstyr.

isolation and culture of rat embryonic neural cells: a quick protocol

Vi beskriver en rask metode for å skille og kultur hippocampus eller kortikale nevroner fra E15-17 rotte embryoer. Prosedyren kan brukes med hell til isolering av mus og menneskelige primære nerveceller og nevrale stamceller. Dissosiert nevroner er opprettholdt i serum-fri medium opptil flere uker. Disse kulturene kan brukes til nucleofection, immunocytochemistry, nukleinsyrer forberedelse, samt elektrofysiologi. Eldre nevrale kulturer kan også bli tilført med en god virkningsgrad ved lentiviral transduksjon og mindre effektivt, med kalsiumfosfat eller lipid-baserte metoder som lipofectamine.

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Vi beskriver en rask metode for å isolere og kultur hippocampus og kortikale nevroner fra gnagere embryoer. Denne protokollen tillater oss å utføre eksperimenter der nesten rene nevrale kulturer er nødvendig.

Isolering og kultur Rat Embryonale nevrale celler: En rask protokoll

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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