Fv10i w

The level of intracellular ROS was assessed fluorometrically by a dihydroethidium (DHE) approach [60 (link)]. This assay is used to detect active ¹O₂ radicals in cells in the presence of other ROS. A 30 mM DHE solution was prepared by dissolving 10 mg of DHE (95%, Sigma Aldrich, St. Louis, MO, USA) in 1 mL of Dimethyl sulfoxide (Sigma Aldrich, St. Louis, MO, USA). Cells were seeded in glass-bottom Petri dishes to form the BBB. After 24 h, DHE was added to the cocultures in an amount of 1 μL per 1 mL of medium and incubated for 30 min. Control plates were washed with nutrient medium after 30 min and imaged with a confocal microscope (λ_ex = 540 ± 25 nm; λ_em = 600 ± 40 nm) for 10 min (Time Laps). The experimental plates were washed with nutrient medium, irradiated with the 1268 nm laser and immediately examined using a confocal microscope under the same conditions. Confocal microscopy was performed using a fully automated confocal laser scanning microscope with water immersion Olympus FV10i-W (Olympus, Tokyo, Japan). Fluorescence level and quantitative data of fluorescence intensity were calculated by FV10-ASW 4.0 microscopy software (Olympus, Tokyo, Japan).

Immunocytochemical staining was performed as described previously [48 , 52 ]. Briefly, the fixed cells were incubated with mouse anti-IDE (1:300) and/or anti-LC3B (1:300) primary antibodies in PBST (PBS with 0.2 % Triton X-100) buffer overnight at 4 °C. After several washes, the cells were incubated with secondary antibody, and images were taken using a confocal laser scanning microscope (FV10i-w; Olympus).

The damage to tumor cells induced by thermal ablation with MWNTs and NIR irradiation was evaluated in vitro. MCF-7 and MDA-231 cells (1.0 × 10⁴ cells/100 μl) were seeded onto 96-well plates for 24 hours and then incubated with various concentrations of MWNTs for 24 hours. The cells were exposed to irradiation with an 808-nm NIR laser at 5 W/cm² for 0-2 min. Cell survival efficiency was measured using the CCK8 assay as mentioned above at 16 hours post-irradiation.
To further verify the photothermal effect on cancer cells, the cells were stained with Live-Dead cell staining kit (Biovision, Mountain View, CA, US) at 16 hours after the photothermal treatment. Briefly, MCF-7 and MDA-231 cells (1.0 × 10⁴ cells/150 μl) were seeded onto confocal dishes (NEST Biotechnology Co. LTD, Jiangsu, China) and incubated overnight. Then 100 μg/ml MWNTs were added to the culture medium. After 24 h incubation, the cells were exposed to irradiation with an 808-nm NIR laser for 2 min at 5 W/cm² and stained with Live-Dead cell staining kit 16 hours later. Briefly, cells were stained with 0.5 ml staining solution containing 0.5 μl Live-Dye (1 mM) and 0.5 μl PI (2.5 mg/ml). After incubated for 15 min at 37 °C, signals were visualized by confocal microscopy (FV10i-W, Olympus, Tokyo, Japan).