Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging

Background: A highly sensitive, fast, and simple flow cytometric assay to assess human red blood cell (RBCs) viability and aging is reported.

Methods: The assay described in this report is based on the use of acetoxymethyl ester of calcein (calcein-AM), a fluorescein derivative and nonfluorescent vital dye that passively crosses the cell membrane of viable cells and is converted by cytosolic esterases into green fluorescent calcein, which is retained by cells with intact membranes and inactive multidrug resistance protein. The loss of calcein can be easily determined by flow cytometry, and the cytosolic localization of esterases was demonstrated by spectrofluorometric analyses.

Results: We found that RBCs incubated with Ca(2+), which induces a rapid and modulated self-death that shares several features with Apoptosis (Bratosin et al., Cell Death Differ 2001;8:1143-1156), externalized phosphatidylserine and lost calcein staining and cytosolic adenosine triphosphate content. Double labeling using phycoerythrin-labeled annexin-V and calcein-AM showed that the decrease of esterase activity is an early event that precedes the externalization of phosphatidylserine residues. In addition, this assay allowed us to distinguish young and aged RBCs isolated by ultracentrifugation in a self-forming Percoll gradient and can be considered as a reliable marker of RBC aging.

Conclusions: Calcein-AM assay may represent a wide application for assessing RBC viability, particularly in blood banks.