optimize b-catenin topflash assay using wnt3a-CM

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Thu, 10/06/2011 - 21:06
bd@nih
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Joined: 10/06/2011

What cell lines are effective for topflash reporting induced with wnt3a-cm?

What is a reasonable fold increase in transcription under optimal CM and time conditions?

Thanks!

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Mon, 10/10/2011 - 12:38
#1
rnajdi (not verified)
Hello,In my experience, I've

Hello,

In my experience, I've found that HEK293 cells are perfect for reporting TopFlash activity in response to Wnt-conditioned media. I have not tried Wnt3A-conditioned media yet, but Wnt1-conditioned media added for 24h on 293 cells results in about a 50 to a 100-fold increase in reporter activity. It is important to mix the conditioned media with fresh media before adding it to the cells (1:1 ratio). Adding conditioned media alone results in lower activation. I have also tried adding purified Wnt3a (from Stem RD) on 293 cells. In this case, incubation with Wnt3a for 4h results in a 30-fold increase. I hope this helps.

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