Refined annexin V/PI apoptosis protocol to minimize false-positive necrotic cells

Programmed cell death (PCD) is a fundamental biological process required for tissue homeostasis and the removal of damaged or infected cells. Apoptosis, one of the most studied PCD modalities, is routinely assessed using the Annexin V/propidium iodide (PI) assay. However, the conventional Annexin V/PI protocol frequently produces false-positive necrotic signals due to membrane damage caused during cell harvesting and handling. This artifact compromises the metrological accuracy of necrosis quantification, limiting assay standardization in cancer research and potentially leading to the premature exclusion of promising anticancer compounds during in vitro screening. To address this issue, we developed a modified Annexin V/PI protocol designed to improve the reliability of necrosis detection. In the optimized approach, PI is added directly to the culture medium prior to cell harvesting, allowing uptake exclusively in cells with truly compromised membrane integrity. This strategy effectively reduces handling-induced artifacts and provides a more accurate evaluation of the membrane-compromised population. Precise discrimination between apoptotic and necrotic cells is essential for standardized cellular assays and for the accurate interpretation of drug-induced cytotoxic effects. Our revised protocol enhances accuracy, reproducibility, and robustness of apoptosis assessment, particularly in fragile cell systems, while remaining fully compatible with commercially available Annexin V/PI detection kits.