Discrimination of late apoptotic/necrotic cells (type III) by flow cytometry in solid tumors

Abstract

A method is described for the discrimination of Type III, late apoptotic, and necrotic cells, to improve the accuracy of proliferation and ploidy determinations of breast tumors. We selected an immunological probe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, late apoptotic, and necrotic cells. This study utilized MDA-MB-175-VII breast carcinoma cells deprived of oxygen for up to 11 d to simulate intratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means. After 24 h under hypoxic conditions, the MDA cells displayed characteristics associated with both apoptosis and necrosis. Approximately 50% of day 1 cells showed membrane permeability by trypan blue and absence of DNA laddering; however, by day 3-4 characteristic apoptotic DNA laddering by gel electrophoresis was evident. Substantial DNA content loss, further evidenced by a reduction in PI staining and fluorescent microscopy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were positive for antitubulin antibody staining. When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showed a range of 8%-74% with a median value of 11.5%. This study demonstrates that a large percentage of cells in tumors and hypoxic cell populations have significantly reduced DNA content, such that conventional live/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investigations.

Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation

“…Trypan blue uptake is a marker of plasma membrane breakdown, a consequence of cell death. It is certainly a feature of necrosis 41 but has also been observed in apoptotic hepatocytes in culture. 42 It has been suggested that early apoptotic cells maintain exclusion of Trypan blue but that plasma membrane breakdown can supervene during later phases of apoptotic cell death, permitting the penetration of Trypan blue.…”

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

“…Our results would therefore indicate that, in adequate conditions of analysis, flow cytometry of PI-stained, detergent-treated nuclei allows us to detect not only apoptosis, as previously shown (19,20), but also necrosis. Previously, other investigators addressed their interest to the development of flow cytometry-based methods able to discriminate between necrosis and apoptosis (16)(17)(18). To our knowledge, until now all of the methods proposed to distinguish necrosis and apoptosis by flow cytometry were based on entire cell analysis.…”

“…However, these techniques do not allow us to distinguish between nuclei from viable or necrotic cells. For this reason, in the past, other investigators were interested in developing new methods able to detect apoptosis and necrosis within a single sample (16)(17)(18). In the present study, we have focused our attention on setting up the optimal conditions to discriminate by multiparameter flow cytometry the characteristics of viable cells and apoptotic or necrotic dead cells by using a highly reproducible, rapid and easily accessible method.…”

Comparative analysis of apoptosis measured by Hoechst and flow cytometry in non-Hodgkin's lymphomas

“…Morphologically whole apoptotic nuclei have been reported to have a DNA index of 0.1 (33). In addition, late apoptotic cells in solid tumors have been reported to show no PI stainability, although cytoplasmic staining with anti-tubulin antibody is still intact (30).…”