Annexin V for flow cytometric detection of phosphatidylserine expression on lymphoma cells undergoing apoptosis

Abstract

OBJECTIVE: To quantitatively analyze apoptotic and secondary necrotic cells under apoptosis conditions. METHODS: The cells of Burkitt lymphoma cell line Raji were incubated with 1.0 mumol/L dexamethasone (DEX) for 2, 4 and 8 h, then stained with Annexin V-FITC (fluorescein isothiocyanate conjugated) which was used to detect the exposure of phosphatidylserine (PS) on the out membrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows the analysis of secondary necrotic cells related with cell membrane and DNA damage, then apoptotic cells was quantified by flow cytometry (FMC). Furthermore, Annexin+/PI- and Annexin+/PI+ cells were sorted by fluoresence-activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electrophoresis. RESULTS: The results revealed that the percentage of apoptotic cells was increased and correlated well with incubation time (r = 0.97). The sensitivity of this method was shown by its detection limit 0.02%; the method was reproducible, and the coefficient variance (CV) was 4.2%. Meanwhile, the Annexin+/PI- and Annexin+/PI+ cells were identified as apoptotic and necrotic cells under EM, and the DNA extracted from the Annexin+/PI- cells was characterized by "ladder pattern". CONCLUSION: Annexin V assay for analyzing apoptotic cells is specific, sensitive, accurate, reproducible and quantitative for apoptosis investigation.