Figure 4

U87 cells incubated with 1 µM of HPPH formulated in either Tween or cationic PAA-NMe³⁺ and the number of cells was counted utilizing a trypan blue assay. To understand the type of ROS (singlet oxygen or radical ions) responsible of cell-kill mechanism the cells were incubated with carboxy-DCFDA and exposed to US or H₂O₂ (positive control for radical ions) (A) in vitro ROS measurement by Carboxy-DCFDA. Cells were imaged with fluorescent microscope and analyzed using ImageJ Grey Value intensity of the photosensitizer then normalized to number of cells. For detection of single oxygen, SOSG (a singlet oxygen quencher) was added to a test tube along with 10 µM of HPPH and exposed to light or US. The resulting fluorescence of SOSG under the different reactions was measured and graphed (B). To further confirm that SDT does not utilize singlet oxygen, the U87 cells were either grown in normoxia (C) or hypoxia environment (D) with 1 µM of HPPH. (E) Mechanisms for the formation of reactive oxygen species by exciting the PS with either light (Type I) or ultrasound (Type II).