A Comprehensive Tutorial on In Vitro Characterization of New Photosensitizers for Photodynamic Antitumor Therapy and Photodynamic Inactivation of Microorganisms

Table 3

Cell-based assays for discrimination and quantification of cell death modes.

Cellular/biochemical event	Method^a	Assay platform	Comment^b
DNA degradation	(i) Detection of “DNA ladders,” that is, multiples of 185 bp	Gel electrophoresis	Semiquantitative
	(ii) TUNEL	FM, FACS	Semi-quantitative
	(iii) COMET	Single cell gel electrophoresis	Semi-quantitative
	(iv) SubG1 (cell cycle analysis)	FACS	Quantitative
Nuclear fragmentation	For example, DAPI, Hoechst-33342 DNA-stained nuclei	FM	Quantitative
Membrane blebbing	Morphological changes	Phase contrast LM	Semi-quantitative
Caspase activation	Fluorometric/luminometric detection of cleavage of artificial caspase substrates	Microplate reader, FM, FACS	Quantitative, single-cell analysis via FACS
PSer exposure	Antibody staining	FM, FACS	Quantitative, single-cell analysis via FACS
Mitochondrial cyt-c release	Subcellular fractionation and immunodetection	Western blotting	Semi-quantitative
Mitochondrial ΔΨ breakdown	Fluorochrome-based assessment of mitochondrial ΔΨ	FM, FACS	Semi-quantitative (within cells), quantitative for comparison between cell populations
Membrane integrity, release of intracellular material^c	(i) Detection of necrosis-associated plasma membrane breakdown via PI staining	FM, FACS	Quantitative, single-cell analysis via FACS
	(ii) Biochemical assay for LDH enzyme release from necrotic cells	Microplate reader	Quantitative

Cyt-c: cytochrome c; DAPI: 4′,6-diamidino-2-phenylindole; ΔΨ: mitochondrial membrane potential; FACS: fluorescence-activated cell sorter; FM: fluorescence microscopy; LM: light microscopy; PI: propidium iodide; PSer: phosphatidylserine; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
^aSelection of methods is focused on in vitro experimentation (cell culture).
^bBased on the author’s experience.
^cThese methods address specific necrosis-associated cellular changes.