Table 2.
(Immuno)fluorescence microscopy- and immunoblotting-based methods to detect cell death
| Method | Advantages | Drawbacks | Notes |
|---|---|---|---|
| (Immuno)fluorescence microscopy | |||
| AO staining | Allows for the highly specific identification of apoptotic cells | AO undergoes photobleaching after several seconds | The elevated specificity of AO for |
| in live tissues and embryos from various model organisms | Stained tissues must be observed and photographed immediately | apoptotic cells within live tissues and | |
| immediately | embryos is still not fully understood | ||
| AO/EB staining | Very rapid and simple | EB is carcinogenic | AO stains both live and dead cells, while |
| Allows for the discrimination among live, (early and late) | Some expertise may be required to clearly distinguish | EB is taken up only by cells that have lost | |
| apoptotic and necrotic cells | between late apoptotic and necrotic cells | plasma membrane integrity | |
| Calcein retention | Simple technique | Diluted calcein-AM must be used immediately after | Cell-permeant, nonfluorescent calcein- |
| Suitable for both proliferating and nonproliferating cells | preparation, as it spontaneously hydrolyzes | AM is hydrolyzed by IC esterases to | |
| Higher signal-to-noise ratio than other fluorochromes | Calcein is actively extruded by MDR1-overexpressing cells | calcein, which is fluorescent and retained | |
| by viable cells | |||
| Calcein quenching | Allows for the visualization of mitochondria with an intact IM | Reversible permeabilization of the IM leads to the loss of | Detects the loss of barrier function of the |
| Suitable for videomicroscopy | calcein signal in the absence of MPT | IM to ions (in particular to Co2+) | |
| Caspase activation assays | Quantitative analysis on a per-cell basis (as opposed to IB) | Operator dependent | Based on antibodies that recognize active |
| The cleavage of cell-permeant, fluorogenic substrates can | Caspase-activation may occur in cell death-unrelated | caspases or cleaved substrates | |
| be monitored in living cells | settings | Based on cell-permeant fluorogenic | |
| Immunostainings heavily depend on the performance of primary antibodies | substrates | ||
| Δψm-sensitive fluorochromes | Allow for the visualization of energized mitochondria | Δψm can be partially reduced in cell death-unrelated | Cationic lipophilic probes accumulate in |
| •Fixable (e.g., CMXRos) | No need for permeabilization | settings, and this may be hard to differentiate from | mitochondria driven by the Δψm |
| •Nonfixable (e.g., JC-1, TMRM) | Fixable probes may be useful in colocalization experiments | irreversible loss | Ratiometric dyes (e.g., JC-1) change |
| Nonfixable probes allow for real-time monitoring of Δψm | Fixable probes are mitochondrio-toxic and hence suitable | emission spectra as a function of Δψm | |
| only for end-point determinations | |||
| Nuclear counterstaining | Labeling is rapid | Hoechst 33342 and DAPI are very sensitive to | Nuclear pyknosis is a classical hallmark |
| •DAPI | Useful to clearly identify nuclei in colocalization assays | photobleaching Inappropriate on its own to conveniently | of apoptotic cells |
| •Hoechst 33342 | Hoechst 33342 is cell permeant | monitor cell death | |
| Relocalization | No need for subcellular fractionation (as opposed to IB) | Require confocal microscopy | MOMP is monitored by assessing the |
| •IMS proteins (e.g., AIF, Cyt c) | Indicative of the subcellular localization of IMS proteins | At least two IMS proteins should be evaluated, to exclude | subcellular relocalization of IMS proteins |
| •Proapoptotic Bcl-2 proteins | upon mitochondrial release | artifacts | The translocation and full insertion into |
| (e.g., Bax, Bid) | Fusion proteins allow for real-time (video or time-lapse | Two-color colocalization approaches are required (with | the OM of Bax mediates MOMP |
| •Lysosomal proteins | microscopy-based) studies | sessile markers and/or functional dyes specific for other | LMP leads to the cytosolic spillage of |
| (e.g., cathepsins) | organelles) | cathepsins, which are able to induce | |
| Unsuitable for very precise spatial determinations | MMP | ||
| Posttranslational (in)activation | Quantitative analysis on a per-cell basis (as opposed to IB) | Operator dependent | Analysis of structural changes in cell |
| (e.g., Bax, p53) | Detects early biochemical events in cell death cascades | Specific conformations may be unstable and get lost during | death regulators |
| permeabilization or fixation | |||
| TUNEL | Useful in costaining protocols, to confirm DNA | Prone to false-positive results, for instance due to sample | Detection of free 3′-hydroxyl ends in DNA |
| fragmentation | processing | ||
| Immunoblotting | |||
| Caspase activation assays | Applicable to subcellular fractions (as opposed to IF or | Semiquantitative (the analysis involves entire cell | Based on antibodies that recognize active |
| cytofluorometry) | populations) | caspases, their cleaved substrates or | |
| Based on standard laboratory equipment | Small protein fragments (such as degradation products) | both the inactive and active forms of | |
| may be difficult to detect | caspases | ||
| Release of IMS proteins | Allows for the study of subcellular fractions and purified | Time-consuming | MOMP is monitored by assessing the |
| mitochondria (e.g., AIF, Cyt c) | mitochondria (as opposed to IF) | Not suitable for large-scale or high-throughput applications | presence of IMS proteins in |
| May require a significant amount of starting material | nonmitochondrial subcellular fractions | ||
| Posttranslational (in)activation | Allows the monitoring of early biochemical events of the cell | Relies on conformation- or neoepitope-specific antibodies | Analysis of structural changes in cell |
| (e.g., Bax, p53) | death cascade | Specific conformations may be unstable and get lost during | death regulators |
| purification or electrophoresis |
Abbreviations: AIF, apoptosis-inducing factor; AM, acetomethoxy; AO, acridine orange; CMXRos, chloromethyl-X-rosamine; Cyt c, cytochrome c; Δψm, mitochondrial transmembrane potential; DAPI, 4′,6-diamidino-2-phenylindole; EB, ethidium bromide; IB, immunoblotting; IC, intracellular; IF, (immuno)fluorescence microscopy; IM, mitochondrial inner membrane; IMS, mitochondrial intermembrane space; C-1, ′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide; LMP, lysosomal membrane permeabilization; OM, mitochondrial outer membrane; MDR1, multidrug resistance protein 1; MOMP, mitochondrial outer membrane permeabilization; MPT, mitochondria permeability transition; TMRM, tetramethylrhodamine methyl ester; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling