Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems

Abstract

Many parallel processes occur during the final stages of apoptosis. It is not clear which of these processes occur in all or most models of apoptosis and which occur only in some. In addition, the temporal relationship of these events is not always well understood. Correlated flow cytometric measurements were used to address these questions. Several models of apoptosis were studied, including thymocytes treated with dexamethasone, MOLT‐4 cells treated with etoposide, U937 cells treated with anti‐Fas, HL‐60 cells treated with camptothecin, Raji cells grown in low serum, and aged neutrophils. All models showed a decrease in LDS‐751 and fluorescein diacetate (FDA) staining, an increase in staining with dihydrorhodamine 123 (dhR123) or dihydroethidium, and an acidification of the cytoplasm. In each model, these changes were highly correlated, appearing simultaneously as multiparameter measurements. Changes in membrane status detected with merocyanin 540 (MC540) and annexin V behaved differently. A population with LDS‐751 and FDA changes but without annexin V or MC540 changes could be demonstrated in some models. Several models did not show any change in annexin V binding, and HL‐60 did not show a change in MC540 binding during apoptosis. The loss of cell surface antigens (CD45 and CD16) from aged neutrophils occurred in the entire LDS‐751 and FDA dim population, even though other membrane changes (including the appearance of annexin V binding sites) were only apparent in a subset of these cells. These results suggest a model for the ordering of some of the terminal processes in apoptosis, with annexin V and MC540 changes trailing other events in apoptosis. These results confirm the need for caution in using a single‐parameter measurement as an indicator of apoptosis for any new model being studied. Cytometry 28:253–263, 1997. © 1997 Wiley‐Liss, Inc.

Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen-thawed peripheral blood stem cell transplants

“…Our experiments with the KB3-1 cell line confirm the finding of others [12][13][14] that annexin V may fail to detect early apoptosis although at the same time it is able to stain those cells that have permeable membranes. Still, annexin V is potentially able to detect apoptosis earlier than conventional methods as can be demonstrated for CD34 + cells: there is a population of Syto R 16 defined early apoptotic cells that is annexin V positive (Figure 7).…”

“…6,[8][9][10][11] Recently, however, it has been claimed that the annexin V technique may not detect apoptosis in all apoptosis models. [12][13][14] Other plasma membrane changes, eg in antigenic determinants, have been described, suggesting that the plasma membrane may be an early target in the cascade of apoptotic events. [15][16][17][18] A special group of apoptosis markers is represented by the fluorescent vital stains, compounds that passively enter the cell due to their lipophylic character and subsequently stain DNA, RNA, mitochondria or combinations (reviewed in Refs 1-4).…”

Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection

“…The progressive increase of plasma membrane permeability, on the other hand, comes as a typical morphologic alteration of late apoptotic or death cells [32]. This permeability increase allows the entrance and permanence of toxins in the cytoplasm, which in turn, promote the intracellular esterasic activity decrease.…”

“…During HIV infection, both eosinophils and neutrophils might be involved in apoptosis process, promoting the lymphocytes apoptosis rate reduction [32]. Thus, the uses of annexin V-FITC and propidium iodide present limitations that could supply biases in apoptosis determination and quantification which, in turn, must be considered during the choice of sensitive methodologies to evaluate this cellular death process.…”

Replication and cytolysis of an E1B-attenuated adenovirus in drug-resistant ovarian tumour cells is associated with reduced apoptosis

“…16 Both wild-type adenovirus and ONYX-015 are able to induce apoptosis in the chemosensitive A2780 cell line. Neither wild-type nor ONYX-015 are able to induce apoptosis in the drug-resistant A2780/cp70 line.…”