Celleventtm caspase 3 7 green detection reagent

Caspase-3/7 activation was measured after 48 h of incubation with compounds using the CellEvent^TM Caspase-3/7 Green Detection reagent following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). In brief, cells were incubated with compounds for 48 h before the assay. On the day of the assay, the Caspase-3/7 reagent was mixed with complete BrainPhys media to a final concentration of 5 μM and added to cells. After incubation for 30 min at 37 °C, cells were imaged using an ImageXpress Confocal microscope (Molecular Devices, San Jose, CA, USA).

1.5 × 10⁴ (24 h), 1.0 × 10⁴ (48 h) and 6.5 × 10³ (72 h) cells were treated with CAP or argon for 15 s (kINPen) and 30 s (Mini Jet). As positive control cells were treated with cycloheximide (15 µM in cell culture medium; Carl Roth, Karlsruhe, Germany). To be able to normalize the measured fluorescence intensity to the cell number, a second plate was planted out parallel. After the incubation period, the used medium was removed, and 100 μL of Caspase 3/7 detection solution (CellEvent^TM Caspase 3/7 Green Detection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and 10 µM in DPBS with 5% FCS v/v) were incubated for 45 min. The fluorescence (535 nm) was measured after excitation (495 nm) using a plate reader.

Cell viability was determined by staining with Calcein AM (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, 3D spheroids were incubated with Calcein AM staining solution (0.5 µM Calcein AM, 140 mM NaCl, and 20 mM CaCl₂) for 1 h and then washed twice with phosphate-buffered saline (PBS) for 20 min. Dried alginate spots were scanned with an automatic optical fluorescence scanner (ASFA Scanner HE, Medical & Bio Decision). The microscope in the scanner automatically focused on the cell spots by moving in the z-direction, and took 384 individual pictures from a single stained pillar/well plate at 4× magnification. The 384 pictures of the cell spots were then consolidated into a single JPEG image for data analysis. The scanned images were analyzed using CellAnalyzer version 1.0 (Medical & Bio Decision).
Apoptosis was determined by using CellEvent^TM Caspase-3/7 green detection reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, after 3 days of treatment with protons and/or olaparib, spheroids were incubated with caspase-3/7 reagent for 1 h at 37 °C. The fluorescence images were acquired using an ASFA optical fluorescence scanner, and were analyzed using ImageJ software version 1.53e (National Institutes of Health, Bethesda, MD, USA).