Name: quantitative analysis of random migration of cells using time-lapse video microscopy
Uploaded: 2012-05-13T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy at JoVE.com

The authors would like to thank Amanda Struckhoff for the initial help with the experiment. This work was supported by a grant from NIH 5RO1CA115706.

1. Utarbeidelse av Plate Belagt med Collagen <ol><li> Fortynn kollagen til en endelig konsentrasjon på 10 mikrogram / ml i Opti-Mem media. </li><li> Coat 6 vel plater med kollagen, ved å legge 1,5 ml fra trinn 1 til hver brønn. Inkuber over natten ved 4 ° C. </li><li> Dagen etter, vaske tallerkenen 2 ganger med 1x Fosfatbufret saltvann (PBS) og lagre dem i PBS. </li></ol> 2. Celleforberedelsen og Seeding på en seks-brønns plate Merk: Bruk varme ...

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Den sanntid tilfeldig migrasjon analysen muliggjør nøyaktig, følsom, analyse av celle migrasjon parametre som hastighet og forskyvning. Denne metoden er ikke begrenset til slutt punkt måleverdi, derfor er det mer kvantitativ. Siden er celler overvåkes i normal DMEM medium med serum, det reduserer skadelig effekt av lengre inkubasjon i serum frie medier. Det er vist at migrasjon av celler avhenger formatering og bryte celle kontakter med undergrunnen 8, dermed kan denne metoden brukes til å studere effek...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Thermo Scientific</td>
 <td>SH30243.01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Gemini Bio- Products</td>
 <td>100-106</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Opti-Mem</td>
 <td>Invitrogen</td>
 <td>31985-070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagen</td>
 <td>BD</td>
 <td>354231</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope with Live Cell chamber &amp; Automated XY stage controller</td>
 <td>Olympus </td>
 <td>Olympus 1X81</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Environmental control chamber</td>
 <td>Neue</td>
 <td>Neue Live Cell Chamber </td>
 <td>Ours has a custom built rectangular glass plate top for appropriate optics through the top of the chamber. The rectangular shape accommodates multi-well plates used in many of our experiments. </td>
 </tr>
 <tr>
 <td>Automated XY stage</td>
 <td>Prior </td>
 <td>Prior Proscan</td>
 <td>This allows precise return to multiply selected XY positions during time lapse microscopy.</td>
 </tr>
 <tr>
 <td>Camera</td>
 <td>&nbsp;</td>
 <td>Hamamatsu EM camera C9100 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Software</td>
 <td>Typically purchased through microscope vendor such as Olympus</td>
 <td>Slide Book 5 </td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

quantitative analysis of random migration of cells using time-lapse video microscopy

Cell migrasjon er en dynamisk prosess, som er viktig for embryoutvikling, vev reparasjon, immunsystemets funksjon, og tumor invasjon 1, 2. Under retningsbestemt migrasjon, celler bevege seg raskt i respons til en ekstracellulær kjemotaktisk signal, eller som svar på indre pekepinner 3 levert av grunnleggende motility maskiner. Tilfeldig migrasjon oppstår når en celle besitter lav indre retningen, slik at cellene til å utforske sitt nærmiljø. Cell migrasjon er en kompleks prosess, i den første responsen cellen gjennomgår polarisering og strekker utstikk i retning av migrasjon to. Tradisjonelle metoder for å måle migrasjon som Boyden kammeret migrasjon analysen er en enkel metode for å måle chemotaxis in vitro, som tillater måling migrasjon som et sluttpunkt resultat. Imidlertid gir denne tilnærmingen verken måling av individuelle migrasjon parametre, heller ikke det tillate å visualization av morfologiske endringer som cellen gjennomgår i løpet av migrasjon. Her presenterer vi en metode som tillater oss å overvåke trekkende celler i sanntid ved hjelp av video - Time Lapse mikroskopi. Siden cellen migrasjon og invasjon er kjennetegn av kreft, vil denne metoden være aktuelt å studere kreft celle migrasjon og invasjon in vitro. Tilfeldig migrasjon av blodplater har vært ansett som en av parametrene i blodplatefunksjon 4, derfor denne metoden kan også være nyttig å studere blodplater funksjoner. Denne analysen har fordelen av å være rask, pålitelig, reproduserbar, og krever ikke optimalisering av celle tall. For å opprettholde fysiologisk egnede forhold for celler, er mikroskop utstyrt med CO 2 forsyning og temperatur termostat. Cell bevegelse overvåkes ved å ta bilder med et kamera montert på mikroskop med jevne mellomrom. Cell migrasjon kan beregnes ved å måle gjennomsnittlig fart og enverage fortrengning, som er beregnet av Slidebook programvare.

Watch this Scientific Journal Video about Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy at JoVE.com

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Kvantitativ analyse av Random migrasjon av celler ved hjelp av Time-lapse video Mikroskopi

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

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