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Updated: Mar 27, 2026
Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity
Published on: October 28, 2013
Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation
Nikolett Sali1, Sándor Nagy2, Miklós Poór1
1Department of Laboratory Medicine, University of Pécs, H-7624 Pécs, Ifjuság u. 13., Hungary.
Introduction:
In cellular viability assays the sole determination of a single parameter might not give precise information on the extent of toxicity. In our study we worked out a multiparametric microplate assay based on bioluminescent ATP quantification, esterase activity-related fluorescence, nucleic acid staining and total intracellular protein measurement from the same sample in MDCK and HepG2 tissue cultures.
Methods:
Dose-response analyses were done after ATP depletion by metabolic poisons (NaF, NaN3) and by ochratoxin A (OTA) mycotoxin treatments. A novel perchloric acid fixation/extraction technique was applied in order to obtain intracellular ATP levels, esterase activity, DNA content and protein data simultaneously. Esterase activity was assessed by a fluorogenic staining. Estimation of cell number was done by DAPI fluorescence. Our results were expressed as ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein and ATP/DAPI ratios. Apoptosis/necrosis rates were measured by Annexin V-propidium iodide and 7-aminoactinomycin D flow cytometric assays and effects of OTA on actin cytoskeleton were also studied by using labeled phalloidin for visualization of actin.
Results:
We could verify that the esterase assay was not an energy driven (true viability) process. ATP/protein, calcein fluorescence/ATP, calcein fluorescence/protein ratios, DAPI fluorescence and protein levels together with morphological and apoptosis/necrosis parameters deciphered subtle changes in cell viability with good between-run precision. Dose dependent loss in cell number and decreased protein levels were observed in all cases, while disorganization of actin microfilaments was seen in OTA treated cells. The two cell lines did not respond uniformly to the same treatments.
Discussion:
ATP/protein ratio proved to be a useful viability parameter however, the suppression and/or loss of intracellular protein could cause difficulty in interpreting ATP/protein data. We conclude that correct assessment of cellular viability should be done by measuring multiple parameters related to the specific mode of action of the tested toxic compound.
