why need both annexin V and propidium iodide staining for apoptosis - (Mar/03/2008 )

Hi dear all,
I need to check the effect of some drugs on the apoptosis of hepatoma cells. I am pretty new for the apoptosis and Flow cytometry. So I looked some related information but still feel uncomfortable about what I got.

I do not know why I need to stain the cell with both Annexin V and PI for flow cytometry analysis. Why I could not only stained the cells with PI and then check the apoptosis by flow cytometry?

If the Annexin V /PI staining is reasonable, does anybody have a detailed protocol for the flow cytometry analysis(including the cell preparation, Annexin V /PI staining ...)? Do you have any tricks about the adherent cell preparation?

I do appreciate any help from your guys!

Thanks!

The PI is there so you could differentiate between early and late apoptotic cells. When you analyze the cells after flow, populations that are PI+AnnexinV (+) are what you would call "late apoptotic" cells, whereas AnnexinV(+) only cells are early apoptotic (the product info sheet that comes with your vial of Annexin V has pretty good info on this). PI itself is not a good indicator of apoptosis coz necrotic cells can be stained by PI too.

Its always good measures to stain cells with PI whenever I do flow, so I can gate out the dead cells for later analysis

good luck

Hi dear all,
I need to check the effect of some drugs on the apoptosis of hepatoma cells. I am pretty new for the apoptosis and Flow cytometry. So I looked some related information but still feel uncomfortable about what I got.

I do not know why I need to stain the cell with both Annexin V and PI for flow cytometry analysis. Why I could not only stained the cells with PI and then check the apoptosis by flow cytometry?

If the Annexin V /PI staining is reasonable, does anybody have a detailed protocol for the flow cytometry analysis(including the cell preparation, Annexin V /PI staining ...)? Do you have any tricks about the adherent cell preparation?

I do appreciate any help from your guys!

Thanks!

neo blythe, Thank you for your nice reply!

COuld I please know which kit is recommended for Annexin Apoptosis assay?
I am totally new to all of this and cant seem to decide controls n stuff.

If any info, please suggest.

Thanks,

Most of them are okay. I use BD Phamingen or Sigma products. I actually make the Annexin-buffer and PI solution myself as they are easy to make and it saves money.

A good positive control is vital when getting started. Just find a good cytotoxic. Even UVC - if you have a strata-linker handy, can be used. Pour on the cytotoxic, and harvest your cells after 24 hours (give or take - depending on how quickly the cytotoxic works. Make sure you pick a cytotoxic that causes apoptosis - not necrosis.

This way you can be sure your annexin-v staining is working.

Good luck!