Musesoft 1

According to the manufacturer’s instructions, apoptosis was examined using the Annexin V and Dead Cell kit (Millipore). Briefly, after the indicated treatment, the cells were incubated with Annexin V and Dead Cell Reagent (7-AAD) for 20 min at room temperature in the dark. The dead, late-apoptotic, early-apoptotic, and live cell events were counted with the Muse Cell Analyzer (Millipore) and analysed with MuseSoft 1.4.0.0 (Millipore).
Caspase activity was quantified by the Muse MultiCaspase assay kit, which permits the simultaneous detection of the presence of multiple caspases (caspase-1 and 3–9). Briefly, after the indicated treatment, the cells were incubated with Muse MultiCaspase Reagent working solution in 1× Caspase Buffer for 30 min at 37 °C in the dark. The Muse MultiCaspase Reagent contains a derivative VAD-peptide, which binds to activated caspases, resulting in the fluorescent signal proportional to the number of active caspases. Then, the cells were counterstained with Muse Caspase 7-AAD working solution, and the events for necrotic cells, dead cells with caspase activity, viable cells exhibiting caspase activity and viable cells without caspase activity were counted with the Muse Cell Analyzer (Millipore) and analysed with MuseSoft 1.4.0.0 (Millipore).

After the treatment, the cells were washed with phosphate buffered saline (PBS), resuspended in fresh medium, and incubated for 1 h at 37 °C to observe H2AX and ATM phosphorylation, as described previously [45 (link)]. The activation of ATM and H2AX was determined using the Muse^TM Multi-Color DNA Damage kit (Millipore, Hayward, CA, USA) according to the manufacturer's instructions. Following the treatments, the cells were washed with PBS, resuspended in 1× Assay Buffer, mixed with Fixation Buffer (1:1), and allowed to cool. Then, the cells were permeabilized with ice-cold 1× Permeabilization Buffer, resuspended in 1× Assay Buffer, and stained with anti-phospho-ATM (Ser1981)-PE and anti-phospho-Histone H2AX (Ser139)-PECy5 conjugated antibodies for 30 min at room temperature in the dark. The excess of dyes was washed out with ice cold 1× Assay Buffer, and the samples were quantified by Muse Cell Analyzer (Millipore, Hayward, CA, USA). The events for ATM activated cells, H2AX activated cells, cells with DNA double-strand breaks determined as dual activation of both ATM and H2AX, and negative cells, were counted with the Muse Cell Analyzer (Millipore, Hayward, CA, USA) and analyzed with MuseSoft 1.4.0.0 (Millipore, Hayward, CA, USA).