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 til redaktionel assistance. Det beskrevne projekt blev støttet af Award Antal R01MH079751 (PI: F. Peruzzi) fra National Institute of Mental Health. Indholdet er alene forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter fra National Institute of Mental Health eller National Institutes of Health.

1. Poly-D-Lysin (PDL): Fremstilling <ol><li> Tilsæt 5 ml sterilt ddH2O til 5 mg PDL til opnåelse af en stamopløsning af 1 mg / ml. </li><li> Bland stamopløsning ved pipettering flere gange. </li><li> Anvendes straks eller opbevar Poly-D-lysinopløsningen ved 2-8 ° C. </li></ol> 2. Poly-D-Lysin (PDL): Coating plast Cell dyrkningsskåle <ol><li> Fortynd PDL stamopløsningen med sterilt ddH2O til en slutkoncentration på 10 ug / ml. </li><li> Pipetten tilstrækkelig op...

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Fremgangsmåden dissektion og kultur af rotte hippocampale og corticale neuroner beskrevet her tillader udførelse af eksperimenter med næsten rene neuronale kulturer dyrket i et kemisk defineret medium (figur 3). Selv protokoller for dyrkning af næsten rene neuroner i serum-frie medier er tidligere blevet beskrevet 2,3,4, der er vigtige ændringer i vores metode. Forskellig fra traditionelle protokoller (dvs. Banker et al.) 5, har vi erstattet trypsin med TrypLE Express, en mere...

<table border="1"><tbody><tr><td> Reagens </td><td> Koncentration </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> Tabel I. Neurobasal/B27 komplet medium. <table border="1"><tbody><tr><td> Reagens </td><td> Koncentration </td></tr><tr><td> Glucose </td><td> 16 mM </td></tr><tr><td> Saccharose </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 2PO 2 </td><td> 0,22 mM </td></tr><tr><td> Gentamicin </td><td> 50 ug / 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> Osmolariteten </td><td> 320-330 mOsm </td></tr></tbody></table> Tabel II. Dissektionsmedium. <table border="1"><tbody><tr><td> Reagens </td><td> Volumen (pl) </td></tr><tr><td> Neurobasal/B27 komplet medium </td><td> 240 </td></tr><tr><td> Trypan blåsplint 0,4% </td><td> 250 </td></tr><tr><td> Total </td><td> 490 </td></tr></tbody></table> Tabel III. 50x Tælle-opløsning. <table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Kat. nummer </td></tr><tr><td> Dvale 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> Gentamicinsulfat </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 phenolrødt </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Cytosin-β-D-arabinofuranosid hydrochlorid </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poly-D-lysin </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 blåsplint 0,4% </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tabel IV. Specifikke reagenser. <table border="1"><tbody><tr><td> Udstyr </td><td> Firma </td><td> Kat. nummer </td></tr><tr><td> Stereomikroskop </td><td> Olympus </td><td> SZ61 </td></tr><tr><td> Store Tang </td><td> FST </td><td> 11022-14 </td></tr><tr><td> Fin spids tang </td><td> Moria </td><td> MC40B </td></tr><tr><td> Micro fin spids 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 dissekere krum saks </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Glas 2-kammer dias </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> 60 mm skåle </td&gt; <td> BD Falcon </td><td> 353002 </td></tr><tr><td> 100 mm skåle </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 kryo-rør hætteglas </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Tabel V. specifikt udstyr.

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

Vi beskriver en hurtig metode til at dissociere og kultur hippocampus eller cortex neuroner fra E15-17 rotteembryoer. Fremgangsmåden kan anvendes med held til isolering af murine og humane primære neuroner og neurale stamceller. Dissocierede neuroner holdes i serumfrit medium i op til flere uger. Disse kulturer kan anvendes til nucleofection, immunocytokemi, Nucleic Acids præparat, såvel som elektrofysiologi. Ældre neuronale kulturer kan også transficeres med en god virkningsgrad ved lentiviral transduktion og mindre effektivt, med calciumphosphat eller lipid-baserede metoder, såsom lipofectamin.

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

Beskriver vi en hurtig metode til isolering og dyrkning hippocampale og corticale neuroner fra gnavere embryoer. Denne protokol tillader os at udføre eksperimenter, hvor næsten rene neuronale kulturer er påkrævet.

Isolering og dyrkning af Rotte Embryonale Neurale Celler: En hurtig protokol

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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