Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation

Sali, Nikolett; Nagy, Sándor; Poór, Miklós; Kőszegi, Tamás

doi:10.1016/j.vascn.2016.01.004

Cited by 21 publications

(31 citation statements)

References 43 publications

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“…After various treatments, the adherent cell cultures were washed with PBS containing calcium and magnesium, then extraction was performed by 100 µL GEX extraction solution without enzymes and Ampliflu. After 5 min, 10 µL of cell extract was pipetted into white 96-well optical plates containing 100 µL of bioluminescence ATP reagent, and luminescence signal was measured immediately in the multimode reader [ 16 ]. ATP standards were prepared in a separate plate in the range of 17–546 nM ATP.…”

Section: Methodsmentioning

confidence: 99%

“…For the measurement, 20 μL lysate/standard was mixed with 200 μL Bradford reagent. Absorbance was read at 595 nm on the plate reader [ 16 , 55 ].…”

Section: Methodsmentioning

confidence: 99%

“…For appropriate evaluation of ATP levels, results should be referred to a cellular parameter with much more stability than ATP. This might be total cell protein and/or nucleic acid content [ 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

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A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells

Csepregi

Temesfői

Sali

et al. 2018

IJMS

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A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3′-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds.

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Section: Methodsmentioning

confidence: 99%

“…For the measurement, 20 μL lysate/standard was mixed with 200 μL Bradford reagent. Absorbance was read at 595 nm on the plate reader [ 16 , 55 ].…”

Section: Methodsmentioning

confidence: 99%

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A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells

Csepregi

Temesfői

Sali

et al. 2018

IJMS

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“…The resazurin assay and SYBR green I-propidium iodide double-stain fluorescent method enabled us to characterize both the planktonic cell populations and biofilms after the treatment with Ar and Sc. As a limitation of our viability study, it should be mentioned that measuring a single parameter only (e.g., the resazurin assay) does not necessarily reflect total cell viability because cellular ATP levels may change rapidly without significant reduction in intracellular enzyme activities [20]. Based on literature data both Ar and Sc may induce time-dependent cell wall and membrane damage enabling propidium iodide to bind to fungal nucleic acids [21].…”

Section: Discussionmentioning

confidence: 99%

Cytotoxic Action of Artemisinin and Scopoletin on Planktonic Forms and on Biofilms of Candida Species

et al. 2020

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We investigated the antifungal activities of purified plant metabolites artemisinin (Ar) and scopoletin (Sc) including inhibition, effects on metabolic activities, viability, and oxidative stress on planktonic forms and on preformed biofilms of seven Candida species. The characteristic minimum inhibitory concentration (MIC90) of Ar and Sc against Candida species ranged from 21.83–142.1 µg/mL and 67.22–119.4 µg/mL, respectively. Drug concentrations causing ≈10% CFU decrease within 60 min of treatments were also determined (minimum effective concentration, MEC10) using 100-fold higher CFUs than in the case of MIC90 studies. Cytotoxic effects on planktonic and on mature biofilms of Candida species at MEC10 concentrations were further evaluated with fluorescent live/dead discrimination techniques. Candida glabrata, Candida guilliermondii, and Candida parapsilosis were the species most sensitive to Ar and Sc. Ar and Sc were also found to promote the accumulation of intracellular reactive oxygen species (ROS) by increasing oxidative stress at their respective MEC10 concentrations against the tested planktonic Candida species. Ar and Sc possess dose-dependent antifungal action but the underlying mechanism type (fungistatic and fungicidal) is not clear yet. Our data suggest that Ar and Sc found in herbal plants might have potential usage in the fight against Candida biofilms.

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“…Before the treatment, the culture medium was replaced with fresh one (without FBS/HSA, with FBS, or with HSA), then cells were incubated with 50 µmol/L (DHC: 13.313 mg/L; CIT: 12.513 mg/L) or 100 µmol/L (DHC: 26.625 mg/L; CIT: 25.025 mg/L) mycotoxin concentrations in the absence and in the presence of 10% FBS or 40 g/L HSA. After 24-h incubation, ATP levels were quantified applying the previously described method without any modifications(Sali et al, 2016).Statistics Means and standard error (± SEM) values expressed in figures. Statistical evaluation of experiments with site markers and in vitro cell experiments were carried out using one-way ANOVA test (IBM SPSS Statistics, Version 21), during which the level of significance was set at p < 0.05 and p < 0.01.…”

mentioning

confidence: 99%

Interaction of the mycotoxin metabolite dihydrocitrinone with serum albumin

et al. 2018

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Citrinin (CIT) is a nephrotoxic mycotoxin produced by Penicillium, Monascus, and Aspergillus species. CIT appears as a contaminant in cereals, cereal-based products, fruits, nuts, and spices. During the biotransformation of CIT, its major urinary metabolite dihydrocitrinone (DHC) is formed. Albumin interacts with several compounds (including mycotoxins) affecting their tissue distribution and elimination. CIT-albumin interaction is known; however, the complex formation of DHC with albumin has not been reported previously. In this study, we aimed to investigate the interaction of DHC with albumin, employing fluorescence spectroscopy, circular dichroism, and molecular modeling studies. Furthermore, species differences and thermodynamics of the interaction, as well as the effects of albumin on the acute in vitro toxicity of DHC and CIT were also tested. Our main observations/conclusions are as follows: (1) Fluorescence signal of DHC is strongly enhanced by albumin. (2) Formation of DHC-albumin complexes are supported by both fluorescence spectroscopic and circular dichroism studies. (3) DHC forms similarly stable complexes with human albumin (K ~ 10 5 L/mol) as CIT. (4) DHC-albumin interaction did not show significant species differences (tested with human, bovine, porcine, and rat albumins). (5) Based on modeling studies and investigations with site markers, DHC occupies the Heme binding site (subdomain IB) on human albumin. (6) The presence of albumin significantly decreased the acute in vitro cytotoxic effects of both DHC and CIT on MDCK cell line.

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Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation

Cited by 21 publications

References 43 publications

A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells

A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells

Cytotoxic Action of Artemisinin and Scopoletin on Planktonic Forms and on Biofilms of Candida Species

Interaction of the mycotoxin metabolite dihydrocitrinone with serum albumin

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