Review Article

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

Table 1

Tests for cell viability and cell death modes.

Assay typeTestMeasured parameterSignal  InstrumentMicroplateaReferencesb
ABSFILumi

Metabolic enzyme(s)MTT, XTT, WSTMitochondrial enzymesXMicroplate readerYes[7779]
ResazurinCellular dehydrogenase enzymes and cytochromesXMicroplate readerYes[80]

MetabolitesATPIntracellular ATPXMicroplate readerYes[81]

Apoptotic changesCaspase activationApoptosis-specific proteasesXXXWestern blotNo[77]
Microplate readerYes[75]
Nuclear fragmentationChromatin condensation and fragmentationXFluorescence microscopeYes[82]
Flow cytometerNo[75]
DNA ladderDNA cleavage resulting in multiples of 180 BpXGel electrophoresisNo[83]
Membrane blebbingCharacteristic apoptotic bodiesXPhase contrast microscopeYes[67]
PARP cleavagepoly-ADP ribose polymerase cleavageXXWestern blotNo[77]
Annexin VMembrane externalization of Annexin VXFlow cytometerNo[77]
Fluorescence microscopeYes[77]

Cyt-c releaseMitochondrial cyt-c releaseXXWestern blotNo[77]

Mitochondrial membrane potential breakdownXFlow cytometerNo[67, 84]
Fluorescence microscopeYes[85]

Cell proliferation 3H thymidineDNA incorporation of 3H thymidine/BrdU Scintillation counterYes[86]
BrdUXMicroplate readerYes[87, 88]

Cell numberDirect cell numberXFlow cytometerNo

ABS: absorbance; ATP: adenosine-5′-triphosphate; BrdU: 5-bromo-2′-deoxyuridine; caspase: cysteine-dependent aspartate-directed proteases; FI: fluorescence intensity; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Lumi: luminescence; PARP: poly(ADP-ribose) polymerase; WST: water-soluble tetrazolium salt; XTT: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.
aAssay suitable for use with microplates (yes/no).
bMethodological references or exemplary studies using the respective test in the context of in vitro PDT.