Apoptosis and Necrosis Quantification

The quantitative analysis of apoptotic and necrotic dead cells was conducted using the flow cytometer Muse ™ Cell Analyzer (Merck Millipore, Billerica, MA, USA) and the Muse™ Annexin V and Dead Cell Assay Kit (MCH100105; Merck Millipore) according to the manufacturer’s instructions. The mock and irradiated C. reinhardtii cells were harvested at 6, 24, and 48 h after X-irradiation, washed twice with Dulbecco’s PBS, stained with Muse™ Annexin V and Dead Cell Reagent at a final concentration of 5 × 10⁶ cells mL ⁻¹, and finally subjected to the apoptotic cell death assay using 5,000 cells for each sample. Apoptotic cell death was expressed as the proportion of living, early/late apoptotic, and dead cells, which were determined using Muse™ Cell Analyzer software (Muse 1.1.2; Merck Millipore).