Operetta clstm high content imaging system

Single cell morphology analysis was performed using Operetta CLS^TM High Content Imaging system (PerkinElmer, Skovlunde, Denmark) as we described in a previous study^{28 (link)}. Briefly, the cells were trypsinized and seeded as 1000 cell/well into clear bottom 96-well CellCarrier™ microtiter plates (PerkinElmer, Denmark). The cells were fixed in 4% paraformaldehyde for 10 min, washed with PBS, stained with DAPI (Sigma-Aldrich, D8417) and Phalloidin-TRITC (Sigma-Aldrich®, P1951). Fluorescent images were analyzed at × 10 and × 40 magnification using Harmony High Content Imaging and Analysis Software (PerkinElmer, Denmark). All pictures were acquired with the same contrast and brightness parameters, and data analysis performed by same parameters setting.

The high content imaging system was used to visualize autophagic vacuoles within live cells. HA and A172 cells were grown in ViewPlate-96 Black, clear bottom, TC-treated (PerkinElmer, 6005182), and cultured overnight. The treated cells were stained using Cell Meter™ Autophagy Assay Kit (AAT Bioquest, 23002) and Hoechst 33,342 (Meilunbio, MA0126) based on the manufacturer’s instructions. The images were acquired and analyzed through the Operetta CLSTM High content imaging system (PerkinElmer, Waltham, MA, USA).
Confocal microscopy was used to visualize and monitor autophagy flux in live cells. HA and A172 cells were seeded into a 35 mm confocal dish (NEST, 801001) and cultured overnight. Then, the cells were treated with MitoQ for 2 h. After that, cells were treated using the nuclear dye Hoechst 33,342, Autophagy Assay Kit (green), and LysoBrite Red (AAT Bioquest, 22645). The confocal images were acquired through Laser confocal microscopy (LSM700, Zeiss, Germany).