If you collect ~10K events, you should see defined populations that you can either gate or make a quadrant around. There are specific ways of establishing your gates/ quadrants, but for the most part, unless you are using other stains at the same, time, you can simply eyeball it. Please see an example that I clipped from miltenyi

Early apoptotic cells can be detected with the help of annexin V; a protein encoded by the ANXA5 gene, which acts as an anticoagulant. Annexin V, as other members of the annexin protein family, binds to specific membrane proteins. During the early stages of apoptosis the plasma membrane is rearranged, which results in changes to the cell membrane’s phospholipid asymmetry. One of the effects of these rearrangements is the display of phosphatidylserine (PS) at the outer plasma membrane. In healthy, quiescent cells PS is exclusively located at the inner membrane and therefore the exposure of PS is an indication of early apoptosis (Fadeel and Xue, 2009). Annexin V has a high binding affinity for PS in the presence of Ca2+ and is therefore a common tool for detecting apoptotic cells.

https://www.bio-rad-antibodies.com/annexin-v-kit.html

Hello Nawal

In addition to the above answers just go through the link below. All your doubts may get cleared.

https://voices.uchicago.edu/ucflow/2012/07/08/my-3-step-approach-to-gating-annexin-v-data-appropriately/

Best Wishes.

Thank you all for your generous answers.

My questions were about:

1- In the attached image (1), there's 2 plots shows that the map was moved to the corner next to the red arrow and the other plot is far from the corner, why? when do I move the contour map and based on what?

2- From the attached image (2), how do I distinguish the depris from the live cells?

3- In the attached image (3), why is the live cell square is bigger than necrotic cells? based on what do I decide?

Here you go.