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Why must Annexin V–PI assays be analyzed by flow cytometry within 1 hour after staining?
Because apoptosis is a rapid process, it is recommended to analyze samples within one hour after staining. PI is highly time‑dependent; labeling with PI increases cytotoxicity, particularly when detecting early apoptosis. Prolonged incubation not only widens the separation of cell populations on the flow cytometer but also significantly amplifies measurement errors; therefore, analysis should be performed within one hour.
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