Green Fluorescent Protein-Based Viability Assay in a Multiparametric Configuration
| Autor: | Miklós Poór, Rita Csepregi, Viktória Temesfői, Tamás Kőszegi, Zsuzsanna Faust |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
green fluorescent protein Programmed cell death Cell Survival Green Fluorescent Proteins Pharmaceutical Science Apoptosis Cycloheximide Article Analytical Chemistry Green fluorescent protein Flow cytometry lcsh:QD241-441 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Adenosine Triphosphate lcsh:Organic chemistry Drug Discovery Sodium fluoride medicine Humans Propidium iodide Viability assay Physical and Theoretical Chemistry cell viability medicine.diagnostic_test flow cytometry fungi Organic Chemistry microplate assay Molecular biology Ochratoxins Luminescent Proteins 030104 developmental biology chemistry Chemistry (miscellaneous) A549 Cells 030220 oncology & carcinogenesis Molecular Medicine Sodium Fluoride Propidium |
| Zdroj: | Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry Molecules, Vol 23, Iss 7, p 1575 (2018) Molecules Volume 23 Issue 7 |
| ISSN: | 1420-3049 |
| Popis: | Green fluorescent protein (GFP) is considered to be suitable for cell viability testing. In our study, GFP transfected A549 lung carcinoma cell line was treated with sodium fluoride (NaF), cycloheximide (CHX) and ochratoxin A (OTA). GFP fluorescence, intracellular ATP, nucleic acid and protein contents were quantified by a luminescence microplate assay developed in our laboratory. Flow cytometry was used to confirm the findings and to assess the intensity of GFP during different types of cell death. A 24 h NaF and CHX exposure caused a dramatic decrease in ATP contents (p < 0.05) compared with those of the controls. GFP fluorescence of the cells was in close correlation with total protein however, GFP/ATP increased at NaF and decreased at CHX treatments (p < 0.05). ATP/protein and ATP/propidium iodide (PI) were largely decreased at NaF exposure in a dose-dependent manner (p < 0.05), while CHX and OTA showed markedly fewer effects. Both treatments caused apoptosis/necrosis at different rates. NaF induced mainly late apoptosis while OTA, mainly apoptosis. CHX effects varied by the incubation time with 100-fold elevation in late apoptotic cells at 24 h treatment. GFP intensity did not show a significant difference between live and apoptotic populations. Our results suggest when using GFP, a multiparametric assay is necessary for more precise interpretation of cell viability. |
| Databáze: | OpenAIRE |
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