methods for detection of apoptotic cells
1. DNA fragmentation assay (sub-G1 peak) using propidium iodide:
- EtOH fixation (small fragments of DNA going out of cells, less DNA = sub-G1;
formaldehyde would retain the fragments in the cell = no sub-G1 visible)
- DNA stainings: PI, Hoechst, DAPI
2. Changes in the cell membrane
A. Annexin V binding to the phosphatydylserine (PS) residues which are externalized during apoptosis
B. Cell membrane becomes permeable to YO-PRO-1 (FITC detector). This DNA dye enters compromised membranes before PI.
3. Change in mitochondrial potential
TMRE (tetramethylrohodamine ethyl ester) accumulates in active mitochondia; during apoptosis the mitochondrial membrane potential is lost so less dye accumulates (PE detector). To read more go to the Mitochondrial membrane potential section.
To distinguish between dead cells and cells during the process of apoprosis use one of following combinations:
- AnnexinV + PI
- YO-PRO-1 + DRAQ7 or
- TMRE + DRAQ7 (APC detector)
4. Detection of enzyme activation
Caspases are important in the execution of apoptosis. Caspase-3 is particularly relevant. One can detect it via specific antibody or enzyme activity kit, such as PhiPhiLux (specific sequence for the enzyme) or FLICA (fluorochrome-labelled inhibitors of caspase activity).
5. Detection of changes to DNA
To monitor late apoptosis, apart from DNA fragmentation assay, detecting sub-G1 peak, which is informative and cheap, one can apply any method mentioned in the DNA damage section. During late stage of apoptosis, specific endonucleases act on the DNA. We can detect this by:
A. looking for strand breaks in DNA (TUNEL) or
B. staining for γH2AX or PARP.