Comparison of Annexin V and Calcein-AM as Early Vital Markers of Apoptosis in Adherent Cells by Confocal Laser Microscopy

Abstract

SUMMARYAlthough morphological criteria for apoptosis are in general reliable, no systematic comparison of the techniques employed thus far has yet been performed. In this study, using confocal laser microscopy, we compared the performance of annexin V-FITC and calcein-AM for early detection of apoptosis in living adherent cells. Experiments were carried out on two distinct cell lines, PC 12 and NIH3T3, endowed with different shape and adhesion properties. The apoptotic process was followed for a prolonged period in the same cells of a predetermined field by means of a special flow chamber. Our results show that both probes allowed the detection of apoptotic cells in either cell line. However, some cells that clearly exhibited apoptotic changes on calcein visualization were annexin-negative. In NIH3T3 cells, annexin negativity of apoptotic cells was correlated with the preservation of cell shape and adhesion properties. These findings show that, at least in PC12 and NIH3T3 cells, annexin might be less sensitive than calcein-AM for early apoptosis detection and, for NIH3T3 cells, suggest that phosphatidilserine exposure is in some way linked to changes in cell shape and/or adhesion to culture substrate.

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

“…When plasma membrane becomes compromised, esterase activity ceases and ethidium homodimer (EthD-1) can react with nucleic acids, producing red fluorescence [19]. Thus, calcein-AM allows determination of cellular viability through esterase activity decrease, while EthD-1 allows verifying the physical and chemical alterations in the cellular membrane, with elevation of 40 times in its fluorescence when associated to nucleic acids.…”

“…Thus, calcein-AM allows determination of cellular viability through esterase activity decrease, while EthD-1 allows verifying the physical and chemical alterations in the cellular membrane, with elevation of 40 times in its fluorescence when associated to nucleic acids. In previous studies, calcein-AM proved to be specific and sensitive for detection and tracking of apoptosis in living cells, as PC 12 and NIH3T3 [19,20]. Considering the fact that these studies were performed with adherent cells and did not include mononuclear cells, such as lymphocytes, the present study compared the performances of calcein-AM and ethidium homodimer (EthD-1) on apoptosis and cellular viability quantification and detection from peripheral blood mononuclear HIV-infected cells, maintained in RPMI1640 during 24 and 48 hours, with annexin V-FITC and propidium iodide, which is widely employed in cytometry as an early marker for apoptosis.…”

Early phase of amyloid β42-induced cytotoxicity in neuronal cells is associated with vacuole formation and enhancement of exocytosis

“…Calcein-AM is permeable for cell membrane and is converted by intracellular esterase into an impermeant fluorescent analogue, calcein (Gatti et al, 1998). EthD-1, another component of calcein-AM assay, enters cells with damaged membranes, binds to nucleic acids and produces a bright red fluorescence in dead cells.…”