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Problems for MTT in SH-SY5Y cell line


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#1 yean_ny_nie

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Posted 10 December 2009 - 06:29 PM

Dear all,

I'm doing a MTT assay for my cell line, SH-SY5Y to check for its viability through different transfected alpha-synuclein mutants. Below is a summary of the methods I've used.

1. First day: Plate cell to 20,000cells/well in 96-well plate.
2. Second day: Change the medium from DMEM to OPTI-MEM.
3. Third day: Add MTT.

The problems arises after I've added the MTT. After 4 hours of incubation with MTT reagent, there is no purple precipitations form. Hence I elongate the incubation time to 24hrs as what I've read from some guideline, different cell line might need different incubation time for the MTT to react.

However, after 24hrs, there is still no purple precipitation. I need help on this. My lab mate which is using the same MTT reagent as me for different cell line have no problems in doing this assay.

I would like to know if there is anyone has encounter any problem with this cell line bfore while doing the MTT? Could it be the seeding density problem? for your information, my junior is handling the same cell line as me and they too have the same problems. Please help!

Thanks for every feedback n reply. It is very much appreciated.

#2 bob1

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Posted 20 December 2009 - 04:09 PM

Don't change the medium, you shouldn't need to as far as I can tell. It could well be affecting the results you get from the MTT as cells don't just automatically function well in a different medium. If you must use opti-MEM, then wean all your cells onto it and then perform your experiments.

I would try a higher seeding density. The SH-SY5Y line is quite slow growing in my experience.




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