Assays of apoptosis and their relationship to events of apoptosis
| Event . | Assays . | Detection . |
|---|---|---|
| Changes in nuclear morphology: | DNA stains (DAPI) | Microscopy |
| Chromatin condensation, segmentation, | ||
| and formation of apoptotic bodies | ||
| Changes in membrane permeability | Vital dyes (PI) | Microscopy |
| Permeable DNA stains: (DAPI, Hoechst 33258) | Flow cytometry with simultaneous size determination | |
| Changes in membrane composition: | Annexin V binding | Flow cytometry |
| Externalization of phosphatidylserine | Confocal and epifluorescence microscopy | |
| Cleavage of nuclear proteins | Poly ADP ribose polymerase | Western blot |
| Mitochondrial function and integrity | ||
| Changes in permeability transition (ΔΨm) | Vital dyes (DiOC6, JC-1) | Flow cytometry |
| Accessibility to mitochondrial antigens | Apo 2,7 antibody | Flow cytometry |
| Release of cytochrome-c | Anti–cytochrome-c antibody | Flow cytometry, Western blot |
| Production of free radicals | DPPP/dihydroethidium | Flow cytometry |
| Caspase activation | ||
| Detection of caspase cleavage product | Known caspase substrates; PARP, caspase 3, caspase 8, DNA-PK, PK-C | Western blot |
| Detection of active caspase | Anti–activated caspase 3 antibody | Western blot |
| Detection of caspase activity | Cleavage of fluorescent or colorimetric substrate(s) | Fluorometer, plate reader |
| DNA degradation | ||
| Large fragments | DNA stains (EtBr, SYBR green) | Pulse-field gel electrophoresis |
| DNA stains (EtBr) | Comet | |
| Radioactivity (C14) | Detection of radio-labeled DNA by filter binding | |
| Small fragments | DNA stains (EtBr) | Agarose gel electrophoresis (DNA ladder) |
| Radioactivity (C14) | Detection of radio-labeled DNA by filter binding | |
| Sub-G1 peak detection | DNA stains (PI, Hoechst) | Flow cytometry |
| Detection of DNA strand breaks | Terminal dUTP nick end labeling (TUNEL) | In situ hybridization |
| Flow cytometry | ||
| Ligation-mediated polymerase chain reaction | Agarose or polyacrylamide gel electrophoresis |
| Event . | Assays . | Detection . |
|---|---|---|
| Changes in nuclear morphology: | DNA stains (DAPI) | Microscopy |
| Chromatin condensation, segmentation, | ||
| and formation of apoptotic bodies | ||
| Changes in membrane permeability | Vital dyes (PI) | Microscopy |
| Permeable DNA stains: (DAPI, Hoechst 33258) | Flow cytometry with simultaneous size determination | |
| Changes in membrane composition: | Annexin V binding | Flow cytometry |
| Externalization of phosphatidylserine | Confocal and epifluorescence microscopy | |
| Cleavage of nuclear proteins | Poly ADP ribose polymerase | Western blot |
| Mitochondrial function and integrity | ||
| Changes in permeability transition (ΔΨm) | Vital dyes (DiOC6, JC-1) | Flow cytometry |
| Accessibility to mitochondrial antigens | Apo 2,7 antibody | Flow cytometry |
| Release of cytochrome-c | Anti–cytochrome-c antibody | Flow cytometry, Western blot |
| Production of free radicals | DPPP/dihydroethidium | Flow cytometry |
| Caspase activation | ||
| Detection of caspase cleavage product | Known caspase substrates; PARP, caspase 3, caspase 8, DNA-PK, PK-C | Western blot |
| Detection of active caspase | Anti–activated caspase 3 antibody | Western blot |
| Detection of caspase activity | Cleavage of fluorescent or colorimetric substrate(s) | Fluorometer, plate reader |
| DNA degradation | ||
| Large fragments | DNA stains (EtBr, SYBR green) | Pulse-field gel electrophoresis |
| DNA stains (EtBr) | Comet | |
| Radioactivity (C14) | Detection of radio-labeled DNA by filter binding | |
| Small fragments | DNA stains (EtBr) | Agarose gel electrophoresis (DNA ladder) |
| Radioactivity (C14) | Detection of radio-labeled DNA by filter binding | |
| Sub-G1 peak detection | DNA stains (PI, Hoechst) | Flow cytometry |
| Detection of DNA strand breaks | Terminal dUTP nick end labeling (TUNEL) | In situ hybridization |
| Flow cytometry | ||
| Ligation-mediated polymerase chain reaction | Agarose or polyacrylamide gel electrophoresis |