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NFAT-SEAP reporter troubleshooting

SEAP assay Calcium signaling Reporter assay ionomycin cell culture

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#1 martywong

martywong

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Posted 31 July 2012 - 05:07 PM

Hi,
I have been trying to setup a reporter assay for Calcium signaling in a cell culture model. I got Clontech Pathway Profiling Kit and use NFAT-SEAP reporter to transfect HEK 293 and Jurkat cells using Lipofectamine 2000. The transfection efficiency is good in HEK 293 but moderate in Jurkat but background SEAP activity can be measured in both cases using Roche SEAP assay kit. The SEAP assay work perfectly if I use CRE-SEAP and treatment with forskolin, which mean the cells and SEAP are both working. I used ionomycin + PMA to stimulate NFAT-SEAP transfected cells but I could not get any stimulatory signals. The treatment always lowers the SEAP activity, at a dose-dependent way. I also try other NFAT stimulators such as carbachol and TNF-alpha, but none of these work as well.
Any ideas, experience, tricks would be greatly appreciated!





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