In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues

Méry, Benoite; Guy, Jean-Baptiste; Vallard, Alexis; Espenel, Sophie; Ardail, Dominique; Rodriguez-Lafrasse, Claire; Rancoule, Chloé; Magné, Nicolas

Vital dyes, which are fluorescent or colored molecules that distinguish between living and dead cells, provide conclusive tests to assess cell death in vitro. The vital dyes most frequently used include exclusion dyes that cannot cross intact plasma membranes, such as 4′,6-diamidino-2-phenylindole or propidium iodide, for instance, and thus only label dead cells. The dye exclusion assay with trypan blue is widely used in routine laboratory work. Blue-stained cells are dead cells and the percentage of viable cells is calculated as ratio of viable and total number of enumerated cells. Hemocytometer and classic light microscopes are commonly used for cell counting. Cytofluorometry is routinely used in co-staining protocols, and fluorescence microscopy facilitates the identification of dead cells by visual inspection. 21 By contrast, other dyes such as calcein acetoxymethyl ester (calcein-AM) can be used to selectively label living cells, thanks to their lipophilic properties that allow them to penetrate easily into cells; subsequently, they are hydrolyzed by intracellular esterases with a generation of fluorescent and plasma-membrane-impermeant products which are retained exclusively by living cells. If permeability assays remain a quick and inexpensive method to assess cell death and provide robust artifact-free information, it is unable to discriminate between different cell modes. Furthermore, concerning dyes with lipophilic properties, the enzymatic activity of intracellular esterases may be affected by cell death-unrelated phenomena. Another disadvantage of such permeability assays concerns the issue of cell death with an intact membrane, as most cytotoxic agents are not intracellular; thus, it could underestimate cellular damage. Concerning colony formation assays, it is based on the number of cells which form colonies in vitro. Cells are seeded at low densities, and the number of colonies is counted after a growth period. Clonogenic assays constitute the most reliable method for assessing cell viability. However, it tends to become harder and time-consuming when the experiment implies several samples. 22 It is also used to assess cell proliferation despite the fact that the measurement of DNA synthesis is often preferred for studying proliferation as it is easier. Indeed, the method involves labeled DNA precursors that are introduced into cells before the measurement of DNA incorporation, after incubation. The occurrence of cell division in vitro is associated with DNA incorporation through a proportional relationship.