I performed annexin v assay to assess cell death in several cell lines (HEK293, HepG2 and CaCo2) after exposing to Fe3O4 nanoparticles. But, I observed high fluorescence (starting from 10^3- 10^4) even for the unstained sample (no annexin V and PI staining).
Did you tested nanoparticles alone in the media, can it happen that they produce non specific fluorescence in the fluorescent channel you are using for Annexin?
Anton Lennikov thanks for your comment. I tested cells alone, which do not include nanoparticles, as you can see in the images as "unstained". But they also gave high fluorescence in these channels.
Try to change the chromophore from FITC and PE to Cy5 and Cy7. I also agree with Sudeep Kumar Maurya that your gain voltages might be too high for 10*7 fluorescence. Try to get 10*2 by reducing the voltages. These cell lines you use are not highly autofluorescent and easy to perform flow on.
High fluorescence in flow cytometry for an Annexin V assay typically indicates a high level of phosphatidylserine exposure on the outer surface of cells, suggesting early stages of apoptosis or cell death
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