Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Abstract

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Figure 1

Methods to detect cell death-related variables. Nowadays, a cornucopia of techniques is available to monitor cell death-related parameters. Within this ‘methodological abundance/redundancy’, the choice of the most appropriate techniques and the correct interpretation of results are critical for the success of any study dealing with cell death. Here, the most common procedures to detect dead/dying cells are indicated, together with the technical platforms that are required for their execution and the types of specimens on which they can be applied. Please see the main text for further details. Δψ_m, mitochondrial transmembrane potential; HPLC, high-pressure liquid chromatography; MOMP, mitochondrial outer membrane permeabilization; MPT, mitochondrial permeability transition; MS, mass spectrometry; NMR, nuclear magnetic resonance; PS, phosphatidylserine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis