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THP-1 stimulation/collection - (Nov/04/2012 )

Hello,

I'm attempting to establish a protocol with THP-1 cells stimulated with 1uM PMA for 72 hours (I have also being attempting 48hr) then collecting the cells via scraping and staining them for flow cytometry. However, whenever I collect the cells then try trypan blue counting - I discovered most of the cells to be dead. Visually, they look ok under the microscope before scraping, so I'm wondering if this might be the reason they are dying? (Though I have not previously heard of this before...)
Thoughts?

Thanks.

-#Tobias-

100 Nano Gram/ml of PMA is enough for differentiation of THP-1.

-memari-

1uM is also enough (60ng/ml), but I have also tried with 100ng/ml - same issue encountered.

-#Tobias-