Cytation 3 cell imaging multi mode reader system

HeLa cells were transfected with empty vector and mtKR plasmids for 30 h and exposed to visible light for 10, 30, and 60 min, respectively, then at 10, 30, and 60 min after exposure, 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, St. Louis, MO, USA) was added into the cells. Finally, the mean fluorescence intensity (MFI) was detected by Cytation™ 3 Cell Imaging Multi-Mode Reader System (BioTek, Winooski, Vermont, USA). There were 6 replicate wells per group, and the experiment was performed in triplicate.

2,7-Dichlorodihydroflurescein diacetate (DCFH-DA, Sigma-Aldrich) is oxidized to highly fluorescent dichlorofluorescein (DCF) by ROS and was used to assay intracellular ROS production. After 24 h of incubation, transfected and nontransfected cells were plated in 96-well plates (6 × 10³ cells/well) and exposed to 4 Gy X-irradiation. Six, 12, and 24 h after irradiation, cells were incubated in PBS containing 8 μM DCF-DA for 15 min at 37°C and were washed twice with PBS, and then the fluorescence was read by a Cytation-3 Cell Imaging Multi-ModeReader System (BioTek, Winooski, Vermont, USA). There were 5 replicate wells per assay, and the assays were performed in triplicate.

MCF-7 and HeLa cells were treated with 1 mM NAC and IR and stained with DCFH-DA (10 μM), and ROS contents were analyzed by Flow Cytometry (FCM, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Additionally, HeLa cells were transfected with empty vector and Sarm1-mtKR plasmids for 30 h and exposed to visible light for 10, 30, and 60 min, respectively; then, at 10, 30, and 60 min after exposure, DCFH-DA was added into cells. Finally, the mean fluorescence intensity (MFI) was detected by the Cytation™ 3 Cell Imaging Multi-Mode Reader System (BioTek, Winooski, Vermont, USA). The experiment was performed in triplicate.