2.5. Measurement of intracellular ROS
RAW 264.7 cells (1 106 cells) were seeded in a glass bottom dish (MatTek
Corp.) and incubated for 24 h. Cells were treated with HBA, HPOX nanoparticles and
POX nanoparticles for 4 h and incubated with H2O2 (200 mM) or PMA (0.5 mg) for
4 h. DCFHeDA (dichlorofluorescinediacetate) was added to each dish and 0.5 h later
the fluorescence images were made with a confocal laser scanning microscope (Carl
Zeiss). To quantify the fluorescent cells, flow cytometry was also performed with
a Flow Cytometry Caliber (Becton Dickinson, US). The percentage of cells in positive
events was calculated as the events within the gate divided by total number of
events then subtracting percentage of the control sample