Harmony 4

In all, 1 × 10⁴ cells were plated in 96-well culture plates (Nunc) in complete DMEM. Sixteen hours later, cells were challenged with indicated drugs for 24 h. Cells were then exposed to plasma (30 s) and further incubated for 6 h. Cells were then fixed in 4 % paraformaldehyde (Sigma) for 30 min. Cells were washed in PBS and counterstained with Hoechst 33258 and cell trace yellow (Thermo Fisher), and imaged with a × 40 water immersion objective using a live cell high throughput imaging system (Operetta CLS; PerkinElmer). Micronuclei were detected and quantified using dedicated image analysis software (Harmony 4.6; PerkinElmer).
For immunofluorescence, cells were blocked in 5% normal serum/0.3% Triton X-100 in PBS for 1 h. Cells were then incubated with anti phospho-ATM and anti phospho-H2AX (γ-H2AX) antibodies (Cell Signaling, 1:1000) diluted in 1% bovine serum albumin/0.3% Triton X-100 in PBS overnight at 4 °C. Cells were washed three times with PBS, followed by incubation with diluted secondary fluorescent antibodies (Alexa Flour 647; 1:5000) for 1 h. After final washing, cells were counterstained with PBS containing Hoechst 33258 (Thermo Fisher) and imaged using a × 40 water objective in high-throughput imaging system (Operetta CLS; PerkinElmer). Image analysis and quantification was performed by dedicated imaging software (Harmony 4.6; PerkinElmer)

The images were obtained using Opera Phenix High Content Screening System and Harmony 4.8 software (PerkinElmer, Waltham, MA, USA) with a 63× water immersion objective (NA 1.15). Alexa 488, Bodipy FL dye-labeled LDL, and pHrodo™ Green conjugate signals were visualized with a 488 nm bandpass excitation filter and 500–550 nm bandpass emission filter. DsRed2 signal was visualized with a 561 nm bandpass excitation filter and a 570–630 nm bandpass emission filter. PureBlu™ Hoechst 33342 signal was visualized with a 405 nm bandpass excitation filter and a 435–480 nm bandpass emission filter. At least thirty 16-bit images were acquired for each sample in confocal mode with a resolution of 1080 × 1080 pixels and binning 2. Exposure time and laser power were kept constant (for each type of imaging) across different repetitions of one type of experiment. Images were processed with Harmony 4.8 software (PerkinElmer). Image analysis to quantify the fluorescence intensities was accomplished using the public domain software, ImageJ 1.53q (NIH, Bethesda, MD, USA).

Caspase 3/7 activity upon TSC treatment was determined using high-content analysis. The bladder cancer cell line J82 was subjected to the same treatment protocol as described for the apoptosis assay. After the incubation with TSC, the medium was aspirated, and the cells were stained with mixture of Hoechst 33342 and CellEvent^TM caspase-3/7 Green Detection Reagent for 30 min. Imaging was conducted using Operetta CLS (PerkinElmer, Waltham, MA) with excitation filters at 386 nm and 488 nm for Hoechst 33342 and the caspase 3/7 Green Detection Reagent, respectively. The proportion of activated caspase 3/7 cells was quantified using Harmony 4.9 high-content imaging and analysis software Harmony 4.9 (PerkinElmer, Waltham, MA, USA).