Multiparametric luminescent cell viability assay in toxicology models

A critical evaluation

Nikolett Sali, Sándor Nagy, Miklós Poór, T. Kőszegi

Research output: Contribution to journalArticle

10 Citations (Scopus)

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: 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.

Original languageEnglish
Pages (from-to)45-54
Number of pages10
JournalJournal of Pharmacological and Toxicological Methods
Volume79
DOIs
Publication statusPublished - May 1 2016

Fingerprint

Toxicology
Assays
Cell Survival
Cells
Adenosine Triphosphate
Fluorescence
Proteins
Esterases
Actins
Actin Cytoskeleton
Necrosis
Cell Count
Apoptosis
Staining and Labeling
Toxic Actions
Phalloidine
Tissue culture
Sodium Azide
Propidium
Mycotoxins

Keywords

  • ATP depletion
  • ATP/protein ratio
  • Cell cultures
  • DAPI staining
  • Luminescence viability assays
  • Perchloric acid extraction

ASJC Scopus subject areas

  • Pharmacology
  • Toxicology

Cite this

Multiparametric luminescent cell viability assay in toxicology models : A critical evaluation. / Sali, Nikolett; Nagy, Sándor; Poór, Miklós; Kőszegi, T.

In: Journal of Pharmacological and Toxicological Methods, Vol. 79, 01.05.2016, p. 45-54.

Research output: Contribution to journalArticle

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