Dear all,

I want to quantify apoptosis in adherent neonatal cardiomyocytes using FACS and PI (the only things available in my lab). How to proceed? The cells seem to be very fragile to tripsin. Can I use Triton x-100? With Triton, what is the particle crossing the laser facs, isolated nucleus or whole permeabilized cells? Sorry, english is not my native language and I need help from anywhere. Thanks a lot.

Propidium iodide (PI) will only tell you if your cells are dead but won't allow you say if they died by necrosis or apoptosis. You need the Annexin V-FITC to do that.

I'm not sure why you want to use Triton X100. This treatment will permeabilise the cells and they will all be dead (PI positive) even thought they weren't.

There's another post on this forum either in cell biology or immunology about annexin V staining and how to neutralise trypsin using a wash with FCS. This may solve or at least limit your problem with trypsin affecting your cells viability.

Ideally you could do some biochemical assay (using a caspase substrate assay) which would allow you not to have to use trypsin.

Have you got access to a fluorescence microsope? Maybe you could permeabilise the cells, stain with PI and look for membrane blebbing? Or look for DNA laddering on agarose gels?

Ceri

May use a cell scraper to lift the neonatal cardiomyocytes.

Hope this may help.