P21493

Necrosis in vitro was induced by stimulation of oxidative stress with H₂O₂ or calcium overload with ionomycin. Plasma membrane permeabilization was determined by PI exclusion and LDH release assays. In brief, cardiomyocytes were incubated with PI (P21493, Thermo Fisher, 2 μg/ml) for 30 min at 37 °C. Necrotic cells (PI⁺) were examined under a fluorescent microscope. LDH assay was performed using the Cytotoxicity Detection Kit (LDH) (11644793001, Roche) according to the manufacturer's instructions. In addition, cell viability was also assessed using Cell Proliferation Kit I (MTT, Roche). Both LDH and MTT assays were measured with Synergy NEO microplate reader (Biotek).

Leaves were fixed in FAA (4% formaldehyde, 5% glacial acetic acid, 50% ethanol), vacuum infiltrated, dehydrated through an ethanol series, rehydrated to 100% water, stained in 0.002% propidium iodide (Thermo Fisher, P21493) for 2 h, dehydrated gently to 100% ethanol, and finally cleared in 100% methyl salicylate (Sigma, M6752) for 7 days. Hand-sections were visualized with a Leica SP8 laser scanning confocal microscope using white light laser excitation at 514 nm with a 20X objective.