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How can the Annexin V–PI assay avoid false-positive results?
Do not overseed cells: When seeding cells in the logarithmic growth phase, maintain a cell density no greater than 1 × 10^6 cells/mL. 6 …to prevent apoptosis induced by the cells themselves during culture, rather than by external experimental conditions; avoid over‑digestion, as excessive digestion can lead to phosphatidylserine (PS) exposure on the cell surface, yielding false‑positive results; handle cells with utmost care—excessive force during cell collection may damage the plasma membrane, exposing its phospholipid layer and enabling Annexin V binding, which can result in false positives. Therefore, when working with adherent cells, proceed gently and minimize any artificial mechanical injury.
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