Operetta cls high content cell imaging analysis system

Logarithmic growth phase cancer cells were treated with different concentrations of Re-CDs in 96-well plates for 24 hours. Then, high-content cell imaging analysis was undertaken to visually reflect the anticancer effect of Re-CDs. An apoptosis and necrosis assay kit (Beyotime Biotechnology) was used in the experiment and the staining assay was carried out as follows. Cells were seeded in 96-well plate and treated with different concentrations of Re-CDs. After 24 hours of incubation, the supernatant was removed and cells were washed with cold PBS. Cancer cells were labeled with Hoechst 33342 and propidium iodide (PI) for 30 minutes at 4°C under working concentration. Cells were washed with PBS three times to remove redundant dye liquors. Images were obtained with the Operetta CLS™ high-content cell imaging analysis system (PerkinElmer Inc., Waltham, MA, USA). The 20× objective lens was applied in each treatment condition. Results were represented by over 40 fields per plate, leading to fields overall acquired for each condition. As we tried to investigate the effect of ROS on apoptosis, the antioxidant N-acetyl-l-cysteine (NAC)- and Re-CDs-treated groups were taken as comparison.

HONE1 cells stained with the CellTracker CM-DiI Dye (C7000, Invitrogen) and activated human primary T cells labeled with live-cell fluorescent dye CMFDA (5-chloromethylfluorescein diacetate, C7025, Invitrogen) at the ratio of 1: 10 were cocultured for 3 hours. The Operetta CLS high-content cell imaging analysis system (PerkinElmer) was used to track the fluorescence status of T cells in real time.