Table 1.
Cell death methodology
| Definition | Notes | Methods of detection3-5 |
|---|---|---|
| Molecular or morphological criteria to define dead cells | ||
| Loss of plasma membrane integrity | Plasma membrane has broken down, resulting in the loss of cell’s identity | (IF) Microscopy and/or FACS to assess the exclusion of vital dyes, in vitro |
| Cell fragmentation | The cell (including its nucleus) has undergone complete fragmentation into discrete bodies (usually referred to as apoptotic bodies) | (IF) Microscopy FACS quantification of hypodiploid events (sub-G1 peak) |
| Engulfment by adjacent cells | The corpse or its fragments have been phagocytosed by neighboring cells | (IF) Microscopy FACS colocalization studies |
| Proposed points-of-no return to define dying cells | ||
| Massive activation of caspases | Caspases execute the classic apoptotic program, yet in several instances, caspase-independent death occurs. Moreover, caspases are involved in non-lethal processes including differentiation and activation of cells | Immunoblotting FACS quantification by means of fluorogenic substrates or specific antibodies |
| ΔΨm dissipation | Protracted ΔΨm loss usually precedes MMP and cell death; however, transient dissipation is not always a lethal event | FACS quantification with ΔΨm-sensitive probes Calcein-cobalt technique |
| MMP | Complete MMP results in the liberation of lethal catabolic enzymes or activators of such enzymes. Nonetheless, partial permeabilization may not necessarily lead to cell death | IF colocalization studies Immunoblotting after subcellular fractionation |
| PS exposure | PS exposure on the outer leaflet of the plasma membrane often is an early event of apoptosis, but may be reversible. PS exposure occurs also in T-cell activation, without cell death | FACS quantification of Annexin V binding |
| Operative definition of cell death, in particular in cancer research | ||
| Loss of clonogenic survival | This method does not distinguish cell death from long-lasting or irreversible cell cycle arrest | Clonogenic assays |
Abbreviations: ΔΨm, mitochondrial transmembrane permeabilization; FACS, fluorescence-activated cell sorter; IF, immunofluorescence; MMP, mitochondrial membrane permeabilization; PS, phosphatidylserine