2.15. Cell Viability and Analysis of Cell Death Mechanism

To assess DLD1 viability, a MTT test was performed. Briefly, after cell treatment with BGNPs and 24 h after irradiation, the media were removed from each well and cells were incubated with a 0.5 mg/mL MTT solution for 90 min at 37 °C with 5% CO₂. At the end of the incubation, the MTT solution was removed and formazan salts were dissolved in 100 µL of DMSO. The absorbance was measured at 570 nm using an EnSpire multimode microplate reader (Perkin Elmer, Waltham, MA, USA). To investigate if the recorded cytotoxic effects were triggered by regulated mechanisms, such as the apoptotic cell death, the Guava Nexin Reagent (Merck, Dramstadt, Germany) was used while containing 7-aminoactinomycin (7-AAD) and annexin V-phycoerythrin. Early events in regulated cell death are characterized by the exposure of phosphatidylserine on the cell membrane, whose integrity remains preserved. Exposed phosphatidylserine is detected by the binding protein annexin V. On the contrary, necrotic cells suffer from the loss of membrane integrity, resulting in being permeable to the dye 7-AAD, which is a DNA intercalator. Accordingly, by using the Guava Nexin Reagent it is possible to distinguish three cell populations, i.e., (i) living cells (annexin V −/7-AAD −), (ii) cells undergoing early phases of regulated cell death (annexin V +/7-AAD −), (iii) and necrotic cells (annexin V +/7-AAD +). Cell viability was analyzed at 3, 6, and 24 h post-irradiation. According to manufacturer instructions, 2 × 10⁴ cells were stained with the reagent for 20 min in the dark at room temperature and analyzed via flow cytometry.