Hi Susannah von Hofsten

I am not experienced in flow cytometry but I carried out Annexin test before, so I would like to answer the last question only.

Cells that are positive to PI but not Annexin may be go through necrotic not apoptosis.

Dear Susannah,

I am not that experienced with Annexin V/PI staining, but since you are hesitating in the gating, I would suggest you to use fluorescent-minus one (FMO) controls. This control should help you to determine positive and negative populations.

Basically, your staining protocol should be something like:

1.- Unstained

2.- AnnexinV+PI

3.- Only Annexin V

4.- Only PI

With the last two, you should exclude problems with spread of the data even after compensation.

For more info, please take a look at:

Fluorescence Minus One Controls - Flow Cytometry Guide | Bio-Rad (bio-rad-antibodies.com)

FMO controls - Flow Cytometry - Service Centers - UTHealth

Hello Susannah, Your Annexin V/PI staining profiles look good to me. The profiles indicate the stage of apoptosis/necrosis in your sample in response to challenge.

progressing from Healthy(LL) -> early(LR) -> intermediate(LR) -> late(UR) -> indistinguishable from necrosis. You don't appear to mention the exposure details prior to evaluation of apoptosis eg 8 hr, 18hr, etc? If you are still in doubt of the progression of cells through apoptosis, just set up a time course. Initially only translocation of phosphotidylserine is externally accessible and detected by AnnexinV binding. This is followed by a progressive loss of membrane integrity, detected by the binding of propidium iodide(or other DNA label). The intensity of the DNA labelling correlates with this membrane failure. In the final stage the phosphotidylserine is shed and only DNA labelling remains and mitigates differentiation from necrosis.

Best wishes

Hi, thank you very much for all your answers! I will consider your suggestions :)