Green Fluorescent Protein-Based Viability Assay in a Multiparametric Configuration.

Rita Csepregi,Zsuzsanna Faust,Viktória Temesfői,Tamás Kőszegi,Miklós Poór

doi:10.3390/molecules23071575

Rita Csepregi, Zsuzsanna Faust + Show 3 more

Open Access

https://doi.org/10.3390/molecules23071575

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Journal: Molecules (Basel, Switzerland)	Publication Date: Jun 28, 2018
Citations: 14	License type: CC BY 4.0

Affiliation: University of Pecs

Abstract

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.

Highlights

Cell viability and cytotoxicity tests are frequently used assays, based on the general considerations about cell death
The molecular basis of green fluorescent protein (GFP) tag/protein expression provides us with a picture about the background of how GFP fluorescence can be used in connection with cell viability [2,3,4,5,6,7]
We found a strong correlation between the GFP fluorescence intensity, cell number, and total protein content during treatments and a remarkably significant difference could be detected in the case of NaF exposure influencing adenosine triphosphate (ATP) levels correlated to GFP intensity, cell number and protein content and at 4 h CHX treatment influencing ATP levels correlated to GFP intensity (p < 0.0001)

Summary

Introduction

Cell viability and cytotoxicity tests are frequently used assays, based on the general considerations about cell death. Cytotoxicity can be assessed by detecting the intracellular protein release. Another significant goal in this regard is the application of methods including metabolic parameters as well [1]. The molecular basis of green fluorescent protein (GFP) tag/protein expression provides us with a picture about the background of how GFP fluorescence can be used in connection with cell viability [2,3,4,5,6,7]. Depending on the construct used for transfection, it can be co-expressed with a specific protein, regulated by the promoter of the protein of interest or randomly integrated into the genome with an own promoter sequence. The HIV-derived lentivectors are stably transfected into A549 lung carcinoma cell line under the control of a cytomegalovirus (CMV)

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Conclusion