*2.7. Cell Death Pathways*

Two methods were used to verify the cell death pathway triggered in the CT26 cells and 4T1 cells by PDT-AlPc-NE protocols: (i) fluorescence microscopy (microscope EVOS-FL, Thermo Fisher Scientific InC., Waltham, MA, USA) of cells stained with acridine orange and propidium iodide (AO/PI) at 4 h after the PDT-AlPc-NE protocols described by Kasibhatla et al. [12] and (ii) flow cytometry of cells stained with Alexa Fluor 488-annexin V and propidium iodide (AnV/PI) at 24 h after the PDT-AlPc-NE. Mitoxantrone (1.5 µM) was used as a positive control for ICD and apoptosis. The necrosis-positive control group consisted of cells frozen and thawed three times, as described by Garg et al. [13].
