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

Abstract

Background: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells 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 prostaglandin E2 treatment and heat shock at 60°C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single‐fluorescence analysis or annexin‐V–fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light‐scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high‐dose detergent treatment 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 were unable, as expected, to discriminate among viability, apoptosis, and necrosis, 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 propidium iodide/annexin V staining. Conclusions: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necrosis. Cytometry 35:145–153, 1999. © 1999 Wiley‐Liss, Inc.

Protection by Herpes Simplex Virus Glycoprotein D against Fas-mediated Apoptosis

“…The identification of apoptotic cells was based on the presence of uniformly stained nuclei showing chromatin condensation and nuclear fragmentation. Flow cytometry analysis of isolated nuclei, following staining with propidium iodide, was performed on a FACScan (Becton Dickinson) as described (46). Analysis of DNA fragmentation at the single cell level was carried out using the TUNEL technique, as described (11).…”

Real‐time flow cytometry for the kinetic analysis of oncosis

“…Previous studies employing flow cytometry to characterize cells undergoing oncosis has focused on the use of annexin V binding to externalized PS to such cells (5,11,12,14). DNA content has also been used to show a difference between apoptotic and heat shock treatment at 568C (11).…”

“…Until recently the flow cytometry community has searched for a means to distinguish between cells undergoing apoptosis or oncosis by standard flow cytometry (6,(11)(12)(13)(14). There are a plethora of assays for the study of apoptosis several hours after induction, by measurement of mitochondrial membrane potential, caspases, annexin V binding through to cell death or necrosis as measured by lack of viability and DNA fragmentation.…”

Modulation of apoptosis during HTLV‐1‐mediated immortalization process in vitro

“…Agonist, anti-human Fas mAb (clone CH11, 500 ng/ml; Upstate Biotechnology, Lake Placid, NY) or caspase inhibitors, Z-VAD-FMK and Z-DEVD-FMK (Calbiochem, San Diego, CA) diluted in DMSO (Sigma, St Louis, MO), were added to some replicate cultures during the last 24 hr of incubation time. In all samples, apoptosis was evaluated by flow cytometry analysis of isolated nuclei following DNA staining with propidium iodide, using a method which distinguishes apoptosis from necrosis and viability [Matteucci et al, 1999]. Briefly, cells were lysed and stained with a solution containing 2% Triton X-100, 25 mg/ml propidium iodide, and 0.05% sodium citrate (all from Sigma).…”