Apoptosis induction: Annexin V/MitoTracker red CMXRos staining: (a) Cells were treated as described in Fig. 7. Three hours after PDT, cells were collected, washed and incubated during 30 min with MitoTracker red CMXRos dissolved in DMEM supplemented with 10% FBS plus antibiotics. Then, cells were washed, stained using Annexin V conjugated to FITC and analyzed by flow cytometry. The results of a representative experiment are represented in dot plot graphs of Annexin V vs MitoTracker red CMXRos. Cells undergoing apoptosis start to exteriorize phosphatidylserine and become Annexin V positive (upper right and left panel). The loss of mitochondrial membrane potential (MMP) can be evidenced by the decrease in MitoTracker red CMXRos staining. (b) The mean percentage ± SEM of cells in each quadrant with respect to the total number of cells is represented in the bar graph. (c) Representative fluorescence histograms of total cells stained with MitoTracker red CMXRos. Note that TAZnPc (second row) and ZnPc (third row) plus light induce a shift in the histogram to lower values suggesting a loss of MMP. (d) The mean fluorescence intensity MFI ± SEM of Mitotracker Red CMXRos is represented in the bar graph. ****p < 0.001 with respect to non-irradiated control cells using one way ANOVA with Dunnett´s post-test (e) Photomicrographs of T98G cells treated with TAZnPc (second row) or ZnPc (third row) stained with MitoTracker red CMXRos to show mitochondrial morphology three hours after irradiation with 0 J/cm2 (left), 10 J/cm2 (middle) or 27 J/cm2 (right). Magnifications of boxed areas are shown in the lower right corner of each photomicrograph.