Investigating apoptosis by flow cytometry - (Mar/21/2007 )

Hi,

Can anyone advise on the best way to look at apoptosis ( in mouse splenocyte cultures) using flow cytometry? is Annexin V a good option? any particular care that is needed when looking at apoptosis?

any advice much appreciated,

D.

Annexin V vs PI is a good way to go or you can do 7AAD or Tunnel stainiing

AnnexinV/PI is an excellent way to measure apoptosis by flow cytometry, the problem with PI only is that it doesnt distinguish between cells that are apoptotic, necrotic or which just have mechanical damage to the plasma membrane [which often happens when you over-trypsinise cells or are too rough when handling them]. So cells that are AnnexinV positive and PI positive could be any of these. To definitively call cells apoptotic they should be AnnexinV positive and PI negative as this is unique to early stage of apoptosis.

Yes its expensive but this will depend on how much/often you need to do this, you can also now get anti-phosphatidylserine antibodies you can use instead of AnnexinV, might be cheaper if you need to do lots.

Other main problem with this is that AnnexinV is usually conjugated to FITC, and there is large spectral overlap between FITC and PI so you would probably need high level of compensation to get good discrimination between the red and green channels. But when you get it working well its great.

For adherent cells, you need handle them very gently when harvesting for AnnexinV/PI or the background will be way high, so for your control sample anything under ~5% AnnexinV positive/PI negative is probably fine.

If you gate out anything that isnt AnnexinV positive/PI negative probably you only see ~30% 'apoptotic' even if all the cells are basically dying, so its more accurate to say that this indicates the *rate* of apoptosis at the timepoint youre looking at rather than what %age of the cells ultimately die.

Thanks so much for the info folks - really helpful!

D.

You can titrate the Annexin V to find the minimal concentration without compromising the results.
In our lab, we dilute Annexin V 1:100 depending on the cell line, and still get good results... SAVE $$$$.

Hope this may help.

I hava an additional question:

what is the best way to detect apoptosis in a mammalian cell line cells?

What is the cheapest?