Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry

Background: Methods widely used to detect apoptosis do not allow us to easi ly distinguish between nuclei from viable or necrotic cells. Even if apopto sis and necrosis seem to occur as alternatives at the single cell level, th ey could be present simultaneously in a cell population much more frequentl y than expected. For this reason, attention was focused on attempting to re cognize, by multiparameter flow cytometry, the characteristics of viable ce lls and of apoptotic or necrotic dead cells. Methods: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prost aglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and prop idium iodide staining followed by single-fluorescence analysis or annexin-V -fluorescein isothiocyanate plus propidium iodide staining by using flow cy tometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolat ion of nuclei by hypotonic solution as well as high-dose detergent treatmen t and DNA staining with propidium iodide. Results: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis w ere unable, as expected, to discriminate among viability, apoptosis, and ne crosis, our new method has enabled us to easily identify nuclei from viable , apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after pro pidium iodide/annexin V staining. Conclusions: A highly reproducible, inexpensive, rapid, and easily accessib le method of analysis has been developed for simultaneously detecting apopt osis and necrcosis. (C) 1999 Wiley-Liss, Inc.