Abstract | 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: 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.
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Authors | Nikolett Sali, Sándor Nagy, Miklós Poór, Tamás Kőszegi |
Journal | Journal of pharmacological and toxicological methods
(J Pharmacol Toxicol Methods)
2016 May-Jun
Vol. 79
Pg. 45-54
ISSN: 1873-488X [Electronic] United States |
PMID | 26777595
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Copyright © 2016 Elsevier Inc. All rights reserved. |
Chemical References |
- Annexin A5
- Fluoresceins
- Fluorescent Dyes
- Mycotoxins
- Ochratoxins
- Proteins
- ochratoxin A
- Dactinomycin
- 7-aminoactinomycin D
- Adenosine Triphosphate
- fluorexon
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Topics |
- Actin Cytoskeleton
(metabolism)
- Adenosine Triphosphate
(metabolism)
- Animals
- Annexin A5
(metabolism)
- Apoptosis
(drug effects)
- Cell Line
- Cell Line, Tumor
- Cell Survival
(drug effects)
- Dactinomycin
(analogs & derivatives, metabolism)
- Dogs
- Dose-Response Relationship, Drug
- Fluoresceins
(metabolism)
- Fluorescent Dyes
(metabolism)
- Hep G2 Cells
- Humans
- Madin Darby Canine Kidney Cells
- Models, Biological
- Mycotoxins
(toxicity)
- Ochratoxins
(toxicity)
- Proteins
(metabolism)
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