University of São Paulo; School of Philosophy, Sciences, and Literature of Ribeirão Preto, Chemistry Department, Center of Nanotechnology and Tissue Engineering -Photobiology and Photomedicine Research Group, Ribeirão Preto, São Paulo, 14040-901, Brazil.
Photochem Photobiol Sci. 2020 Jan 22;19(1):40-48. doi: 10.1039/c9pp00415g.
Although the dichlorofluorescein (DCF) assay is widely used to detect the production of UVA-induced ROS, the photostability and phototoxicity of the probe after UVA irradiation remains controversial and the experimental conditions often vary across studies, making it difficult to compare results from different studies. This study aimed to evaluate the suitability of the DCF assay for detection of UVA-induced ROS in human cells after UVA irradiation. Human primary fibroblasts (HPF) and HaCaT cells were loaded with 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) (2, 10, and 50 μM) for 10 and 30 min, before and after exposure to UVA radiation (5-50 J cm-2). Fluorescence was recorded immediately or 30 min after irradiation using three different techniques: microplate reading, flow cytometry, and confocal scanning microscopy. Cell viability was assessed by flow cytometry before and after UVA exposure. A UVA-dose-dependent increase in ROS was observed at 5-50 μM DCFDA, and the magnitude of the fluorescent signal was affected by RPMI medium, as well as DCFDA loading concentration and incubation period. However, higher concentrations of DCFDA compromised the viability of both HaCaT and HPF cells after UVA irradiation. The most sensitive and reliable combination for the ROS assay was pre-incubation with 10 μM DCFDA for 30 min in PBS. Reading the fluorescence 30 min after UVA irradiation diminished the emission signal, as did the DCFDA post-incubation. In conclusion, this single-point DCF assay allowed reproducible and sensitive UVA-induced ROS detection in HaCaT and HPF cells without compromising the cell viability or morphology.
尽管二氯荧光素（DCF）测定法广泛用于检测 UVA 诱导的 ROS 的产生，但探针在 UVA 照射后的光稳定性和光毒性仍然存在争议，并且实验条件在不同的研究中经常变化，使得难以比较来自不同研究的结果。本研究旨在评估 DCF 测定法在 UVA 照射后用于检测人细胞中 UVA 诱导的 ROS 的适用性。将人类原代成纤维细胞（HPF）和 HaCaT 细胞用 2'，7'-二氯二氢荧光素二乙酸酯（DCFDA）（2、10 和 50 μM）负载 10 和 30 分钟，在暴露于 UVA 辐射（5-50 J cm-2）之前和之后。用三种不同的技术：微孔板读数，流式细胞术和共聚焦扫描显微镜，立即或照射后 30 分钟记录荧光。在 UVA 暴露前后通过流式细胞术评估细胞活力。在 5-50 μM DCFDA 下观察到 ROS 呈 UVA 剂量依赖性增加，并且荧光信号的幅度受 RPMI 培养基以及 DCFDA 加载浓度和孵育时间的影响。然而，较高浓度的 DCFDA 会损害 HaCaT 和 HPF 细胞在 UVA 照射后的活力。ROS 测定的最敏感和可靠的组合是在 PBS 中预孵育 10 μM DCFDA 30 分钟。在 UVA 照射后 30 分钟读取荧光会降低发射信号，而 DCFDA 孵育后也会降低发射信号。总之，该单点 DCF 测定法允许在不损害细胞活力或形态的情况下在 HaCaT 和 HPF 细胞中重现性和敏感地检测 UVA 诱导的 ROS。
