AAT Bioquest

What are the best methods to identify necrotic cells?

Posted May 9, 2020


One method useful for identifying necrotic cells is an Annexin V/PI assay. Annexin V stains positive in apoptotic cells, and PI (propidium iodide) stains positive in necrotic cells. While cells that stain Annexin V+/PI+ may be either late apoptotic or necrotic, only necrotic cells stain Annexin V-/PI+.

Another possible method is gel electrophoresis, where DNA fragments from apoptotic cells would demonstrate a ladder pattern, while DNA from necrotic cells would create a smear on the gel instead.

In addition, HMGB1 or LDH (lactate dehydrogenase) release assays can also be performed, since cells undergoing necrosis release HMGB1 and LDH (apoptotic cells do not release these molecules).

Additional resources

Collins, J. A., Schandl, C. A., Young, K. K., Vesely, J., & Willingham, M. C. (1997). Major DNA fragmentation is a late event in apoptosis. Journal of Histochemistry & Cytochemistry, 45(7), 923-934.

Lee, S. Y., Ju, M. K., Jeon, H. M., Jeong, E. K., Lee, Y. J., Kim, C. H., ... & Kang, H. S. (2018). Regulation of tumor progression by programmed necrosis. Oxidative medicine and cellular longevity, 2018.

Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry

Amplite™ Colorimetric D-Lactate Dehydrogenase (LDH) Assay Kit

HMGB1 Antibody

Cell Viability Assays