Fluoview v2

After the H1299 cells adhered in confocal chamber slides, HmN, HmDN, AHmDN or HHmDN was added into the medium with the final concentration of mPPZ 0.5µM. After the incubation for 12hrs, cells were washed by PBS and then incubated with 10μmol/l 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA) solution (S0033, Beyotime, China) for 30mins in serum-free medium in the dark. The illumination (680nm) at a light fluence of 1.5 J/cm² was followed by ROS Assessment. The fluorescence, based on the oxidative conversion of DCFH-DA to dichlorofluorescein (DCF) by ROS, was detected using Olympus FluoViewTM FV1000 laser scanning confocal microscope (Olympus Corp., Tokyo, Japan) with the excitation at 488 nm. All images were analysed by Olympus Fluoview v2.1 software.

The suspended cells (H1299) were seeded into confocal chamber slides (Nest Biotechnology Co. Ltd., Wuxi, China) and incubated at 37°C for 24 hrs. Then, HmN, HmDN, AHmDN or HHmDN were added into the medium with final concentration of mPPZ 0.5µM. After incubation for 12hrs, adherent cells were washed by PBS to remove unbound nanoparticles and then incubated in medium containing DNA fluorescent dye, DAPI (4ʹ6-diamidino-2-phenylindole, 5µg/mL) for 30 mins. After another wash by PBS, fresh medium was added into the confocal chamber slides and the cells were imaged by Olympus FluoViewTM FV1000 laser scanning confocal microscope (Olympus Corp., Tokyo, Japan). The fluorescence of DOX or mPPZ in the cells was excited by an argon–ion laser light (λ=488nm or 633nm, respectively), while the emitted fluorescence was filtered with barrier filters (590/30 nm or 640–700 nm band pass, respectively). The fluorescence of DAPI was excited by diode laser (λ=405nm) and the emitted fluorescence filtered with barrier filters 450/30 nm band pass. All parameters, including the laser line intensity, photometric gain, settings of photo-multiplier tube and filter attenuation, were kept constant throughout the entire imaging experiment. All images were analysed by Olympus Fluoview v2.1 software.