Differentiate THP-1 using PMA, sth went wrong... - (Jul/09/2008 )

Hi, anyone having experience handling THP-1 plz help me here...

This is what i did but THP-1 didnt differentiated

To 1mg of PMA, i added 10ml of DMSO to make a stock solution of 10,000nM. From the stock, i make a 100nM of working media and induced my THP-1. I prepared this without much light. After leaving in the incubator for 1 day w/o light, the THP-1 didn't adhere to the surface. The stock solution is wrapped in aluminum foil and kept at 4 degree celsius. May i know if i did anything wrong?


Anyone have picture of THP-1 differentiated to macrophage? Plz send to my email (ice2200@hotmail.com) or post it here

Thanks!

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I worked with THP-1 and U937. I didn't see anything wrong with your induction. Even at 10 nM PMA they differentiate. You may also try 10nM PMA and 500nM A23187 as stimulant.

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Which passage did u induce your THP-1?

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Hey pal, PMA stocks work better when they are stored at -20 degrees. I had made the same mistake and got results once stored fresh stocks at -20.

Hey and how many days do you maintain THP-1 with 100nM PMA to get complete differentiation? i am having some trouble with it.

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I agree with laila - PMA should be stored at -20oC and it shouldn't be freeze-thawed. We throw away any leftover PMA even after just one freeze-thaw cycle.

I add 50ng/ml PMA to my cells (1:20,000 dilution from a 1mg/ml stock solution) for 18 hours and then I replace the media and leave the cells for a further 48 hurs (total of 72 hours for full differentiation). So maybe you should try leaving them for longer. Although my cells are definitely stuck after 18 hours of PMA treatment

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May i know which passage did u start to differentiate THP-1 ?

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I waited for 1 days but didnt see any differentiation. So i left it for 3 days but still no differentiation. May i know the protocol of preparing PMA ? And i know PMA is light-sensitive. Is there any precautions to take note of ?

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