Name: assaying dna damage in hippocampal neurons using the comet assay
Uploaded: 2012-12-19T13:00:00
Description: Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Dette arbejde blev støttet af IMPACT Award fra Institut for Radiation Oncology, University of Alabama-Birmingham Comprehensive Cancer Center, Fighting Børnecancerfonden, og Gabrielles Angel Foundation (til ESY.).

1. Cellekultur <ol><li> Kultur neuronale celler og behandle dem efter behov. </li><li> Høst celler i 15 ml rør: aspireres medierne, skylles med phosphatpufret saltvand (PBS, calcium-og magnesiumfri), tilsættes trypsin, opsamles i 15 ml rør og neutralisere trypsin med passende serum indeholdende media. </li><li> Spin ved 1.000 x g i 5 min. </li><li> Sug mediet. </li><li> Resuspender cellerne i PBS. </li><li> Spin ved 1.000 x g i 5 min. </li><li> Aspirér medier og Resuspendér celler i frisk PBS. </li><li> Tælle c...

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Den comet assay har den unikke evne til at analysere de enkelte celler. Dette er fordelagtigt i identifikationen af ​​subpopulationer af celler, der viser forskellen reaktion på cytotoksiske midler. Nogle få praktiske begrænsninger skal tages i betragtning. Antallet af celler, som kan evalueres individuelt kan variere afhængigt af individet. Prøvens størrelse skal øges, hvis der er varians i DNA-skader inden for en population. Levedygtig enkelt-cellesuspension er kritisk for dette assay, da fremherskende tils...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> 1,5 ml rør </td><td> Santa Cruz Biotechnology, Inc </td><td> Sc-200271 </td><td></td></tr><tr><td> 10X TBE (Tris-base, borsyre, EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> 15 ml rør </td><td> Fisher Scientific </td><td> 0553851 </td><td></td></tr><tr><td> Agarose </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Aluminiumsfolie </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Beaker </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Centrifuger </td><td> Thermo Scientific </td><td> 75004261PR </td><td></td></tr><tr><td> Comet Assay software </td><td> Comet Assay IV Image Analysis System </td><td></td><td></td></tr><tr><td> Cylinder </td><td> Fisher Scientific </td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15.575 </td><td></td></tr><tr><td> Electrophoresis kammer </td><td> Thermo Scientific </td><td> 09528101 </td><td></td></tr><tr><td> Ethanol </td><td> Fisher Scientific </td><td> A407P4 </td><td></td></tr><tr><td> Fluorescerende mikroskop </td><td> Zeiss Axio Vision </td><td></td><td> Noget fluorescerende mikroskop med grønt filter er tilstrækkeligt </td></tr><tr><td> Hæmacytometer </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> Agarose med lavt smeltepunkt </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Mikroovn </td><td> Sears </td><td></td><td></td></tr><tr><td> Phosphatbufret saltvand, calciumfri, magnesiumfri </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Strømforsyning </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Køleskab </td><td> Sears </td><td></td><td></td></tr><tr><td> Lineal </td><td> Staples </td><td></td><td></td></tr><tr><td> Skub </td><td> Fisher Scientific </td><td> 12550143 </td><td></td></tr><tr><td> Natriumchlorid </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> Natriumhydroxid </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Natriumlaurylsulfat sarcosinat </td><td> Fisher Scientific </td><td> S529 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> Bakke </td><td> Fishendes Videnskabelige </td><td> 15242B </td><td></td></tr><tr><td> Tris-base </td><td> Sigma </td><td> T6066 </td><td></td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> T8787 </td><td></td></tr><tr><td> Trypsin </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> Vortex </td><td> Fisher Scientific </td><td> 02216108 </td><td></td></tr><tr><td> Vandbad </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

En række lægemidler rettet mod de DNA-reparationsmekanismer og inducerer celledrab ved at skabe DNA-beskadigelse. Således har fremgangsmåder til direkte måler DNA-skader blevet omfattende evalueret. Traditionelle metoder er tidskrævende, dyre, ressource-intensive og kræver replikerende celler. I modsætning hertil er comet assay, en enkelt celle gelelektroforese assay, en hurtigere, non-invasiv, billig, direkte og følsomt mål for DNA-skader og reparation. Alle former for DNA-skader samt DNA-reparation kan visualiseres på enkelt celle niveau ved hjælp af dette kraftfulde teknik. Princippet for kometen assay er, at intakt DNA er meget ordnet hvorimod DNA-skader forstyrrer denne organisation. De beskadigede DNA trænger ind i agarosematrix og når den udsættes for et elektrisk felt, det negativt ladede DNA vandrer mod katoden, som er positivt ladet. De store ubeskadigede DNA-strenge er ikke i stand til at migrere langt fra kernen. DNA-beskadigelseskaber mindre DNA-fragmenter, der rejser længere end den intakte DNA. Comet assay, et billedanalyse-software, foranstaltninger og sammenligner den samlede fluorescerende intensitet af DNA&#39;et i kernen med DNA, der er migreret ud af kernen. Fluorescerende signal fra migrerede DNA er proportional med DNA-beskadigelse. Længere lysere DNA hale betyder øget DNA-skader. Nogle af de parametre, der måles, er tail moment, som er et mål for både mængden af ​​DNA og fordeling af DNA i halen, halelængde og procentdel af DNA i halen. Dette assay kan måle DNA-reparation samt da opløsning af DNA-skader betyder reparationen har fundet sted. Grænsen for følsomhed er omkring 50 strengbrud pr diploid pattedyrcelle 1,2. Celler behandles med DNA-beskadigende midler, såsom etoposid, kan anvendes som en positiv kontrol. Således comet assay er en hurtig og effektiv procedure til at måle DNA-skader.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Det comet assay er en effektiv måde til afsløring af enkelt-og dobbelt-streng pauser, herunder alkali-labile sites og DNA-DNA/DNA-protein tværbindinger på DNA i alle celler, herunder hippocampus neuroner. Fremgangsmåden drager fordel af den differentielle vandring af DNA i et elektrisk felt på grund af forskelle i mængden af ​​DNA-beskadigelse.

Undersøqelse DNA-skader i hippocampus neuroner Brug af Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

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