Department of Laboratory Medicine, Medical School, University of Pécs, Ifjúság u. 13, H-7624 Pécs, Hungary.
János Szentágothai Research Center, University of Pécs, Ifjúság u. 20, H-7624 Pécs, Hungary.
Molecules. 2018 Jun 28;23(7):1575. doi: 10.3390/molecules23071575.
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 ( < 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 ( < 0.05). ATP/protein and ATP/propidium iodide (PI) were largely decreased at NaF exposure in a dose-dependent manner ( < 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.
绿色荧光蛋白（GFP）被认为适合用于细胞活力测试。在我们的研究中，用氟化钠（NaF）、环己亚胺（CHX）和赭曲霉素 A（OTA）处理转染 GFP 的 A549 肺癌细胞系。通过我们实验室开发的发光微孔板检测法，对 GFP 荧光、细胞内 ATP、核酸和蛋白质含量进行定量分析。采用流式细胞术对实验结果进行了验证，并评估了不同类型细胞死亡过程中 GFP 的强度。与对照组相比，24 h 的 NaF 和 CHX 处理会导致 ATP 含量显著下降（<0.05）。细胞的 GFP 荧光与总蛋白密切相关；然而，在 NaF 和 CHX 处理时 GFP/ATP 增加（<0.05）。NaF 暴露时，ATP/蛋白和 ATP/碘化丙啶（PI）的水平呈剂量依赖性显著降低（<0.05），而 CHX 和 OTA 的影响较小。两种处理方式均以不同的速率引起细胞凋亡/坏死。NaF 主要诱导晚期凋亡，而 OTA 主要诱导凋亡。CHX 的作用因孵育时间而异，24 h 处理时晚期凋亡细胞增加了 100 倍。活细胞和凋亡细胞群之间 GFP 强度没有显著差异。我们的研究结果表明，在使用 GFP 时，需要进行多参数检测，以便更精确地解释细胞活力。
