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

Dit werk werd ondersteund door de IMPACT Award van de afdeling Radiotherapie van de Universiteit van Alabama-Birmingham Comprehensive Cancer Center, de Fighting Children&#39;s Cancer Foundation, en de Gabrielle&#39;s Angel Foundation (tot ESY.).

1. Cultuur van de Cel <ol><li> Cultuur neuronale cellen en behandelen hen als dat nodig is. </li><li> Oogst cellen in 15 ml buizen: aspiraat media, spoelen met fosfaat gebufferde zoutoplossing (PBS, calcium en magnesium vrij), voeg trypsine in 15 ml buizen en trypsine passende serum bevattend medium te neutraliseren. </li><li> Spin bij 1000 xg gedurende 5 minuten. </li><li> Aspireren media. </li><li> Resuspendeer cellen in PBS. </li><li> Spin bij 1000 xg gedurende 5 minuten. </li><li> Zuig media en resuspendeer cellen...

<ol>
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De komeet assay heeft de unieke capaciteit van het analyseren van individuele cellen. Dit is voordelig in identificatie van subpopulaties van cellen die andere reactie op cytotoxische middelen tonen. Een aantal praktische beperkingen moeten worden gehouden. Het aantal cellen die afzonderlijk kunnen worden geëvalueerd kan variëren afhankelijk van het individu. De steekproefomvang te worden verhoogd indien er variatie in DNA schade binnen een populatie. Levensvatbare enkele celsuspensie noodzakelijk is voor deze assay s...

<table border="1"><tbody><tr><td> Naam van het reagens </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Opmerkingen (optioneel) </td></tr><tr><td> 1,5 ml buisjes </td><td> Santa Cruz Biotechnology, Inc </td><td> Sc-200271 </td><td></td></tr><tr><td> 10X TBE (Tris base, boorzuur, EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> 15 ml tube </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> Aluminiumfolie </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Beker </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Centrifugeer </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> Cilinder </td><td> Fisher Scientific </td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td> Elektroforese kamer </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> Fluorescentiemicroscoop </td><td> Zeiss Axio Vision </td><td></td><td> Tl microscoop met groene filter voldoende </td></tr><tr><td> Hemacytometer </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> Laag smeltpunt agarose </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Magnetronoven </td><td> Sears </td><td></td><td></td></tr><tr><td> Fosfaat gebufferde zoutoplossing, calcium vrij, magnesium free </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Voeding </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Koelkast </td><td> Sears </td><td></td><td></td></tr><tr><td> Heerser </td><td> Staples </td><td></td><td></td></tr><tr><td> Schuif </td><td> Fisher Scientific </td><td> 12550143 </td><td></td></tr><tr><td> Natriumchloride </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> Natriumhydroxide </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Sodium lauryl sarcosinaat </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> Dienblad </td><td> Fishaar wetenschappelijke </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> Trypsine </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> Draaikolk </td><td> Fisher Scientific </td><td> 02216108 </td><td></td></tr><tr><td> Waterbad </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

Een aantal geneesmiddelen gericht op de DNA herstel routes en induceren celdoding door het creëren van DNA schade. Zo zijn processen om rechtstreeks DNA-beschadiging uitgebreid geëvalueerd. Traditionele methoden zijn tijdrovend, duur, arbeidsintensief en vereisen replicerende cellen. In tegenstelling, de comet assay, een cel gelelektroforese assay, een snellere, niet-invasieve, goedkoop, direct en gevoelige meting van DNA schade en herstel. Alle vormen van DNA-schade en DNA-herstel kan worden gevisualiseerd op de enkele cel niveau met behulp van deze krachtige techniek. Het uitgangspunt van de comet assay is dat intact DNA sterk geordende terwijl DNA-schade verstoort deze organisatie. De beschadigde DNA sijpelt in de agarose matrix en wanneer onderworpen aan een elektrisch veld, het negatief geladen DNA migreert naar de kathode die positief geladen is. De grote onbeschadigd DNA strengen kunnen niet ver van de kern migreren. DNA schademaakt kleinere DNA fragmenten die groter bereik dan het intacte DNA. Comet Assay een beeldanalyse software, meet en vergelijkt de totale fluorescentie-intensiteit van de DNA in de kern met DNA dat uit gemigreerd van de kern. Fluorescerend signaal van de gemigreerde DNA evenredig is met DNA schade. Langere helderder DNA staart betekent verhoogde DNA-schade. Enkele parameters die gemeten zijn staart moment dat een maat is voor zowel de hoeveelheid DNA en de verdeling van DNA in de staart, staartlengte en het percentage DNA in de staart. Deze test maakt het mogelijk om DNA repair meten en aangezien resolutie van DNA schade betekent herstel heeft plaatsgevonden. De grens van de gevoeligheid is ongeveer 50 breuken per diploïde zoogdiercellen 1,2. Cellen behandeld met een DNA beschadigende middelen, zoals etoposide, kan worden gebruikt als een positieve controle. Dus de comet assay is een snelle en effectieve procedure DNA schade te meten.

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

De komeet assay is een efficiënte manier voor het opsporen enkel-en dubbel-breuken, met inbegrip van alkali-labiele sites en DNA-DNA/DNA-protein cross-links op het DNA in alle cellen met inbegrip van hippocampale neuronen. De methode maakt gebruik van de differentiële migratie van DNA in een elektrisch veld als gevolg van verschillen in hoeveelheid DNA schade.

Analyseren DNA schade in hippocampale neuronen Met behulp van de 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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