Intracellular ROS Measurement in T. rubrum

Intracellular ROS production after treating T. rubrum ATCC 28189 with 2-chalcone in the dark and 2-chalcone mediated PDT was evaluated using 50 µM H2DCFDA (2.7 dichlorodihydrofluorescein diacetate, Invitrogen) as described by Singulani et al. (2019) (link). This compound is converted to a highly fluorescent 2ʹ, 7ʹ-dichlorofluorescein (DCF) compound after cleavage of its acetate group by intracellular esterases and this compound binds to ROS. As controls, AMB and FLZ treatments were used. The treatment was performed as described apoptosis/necrosis assay section. After the incubation period, the samples were washed, following suspension in 500 µL of PBS, and transferred to cytometer tubes. Then, 1.5 µL of the H2DCFDA solution was added with subsequent incubation at 25°C in the dark for 10 minutes. The samples were analyzed on a BD FACS Canto I flow cytometer.