Jc 1 kit

HGC-27 cells were pretreated with HMON@CuS/Gd, with or without an 808 nm laser at a power density of 0.8 W/cm² for 5 min respectively. 24 h later, the treated cells were collected and incubated with CCK-8 kit for viabilities detection, with Annexin V-FITC/PI Kit (KeyGEN, Nanjing, China) for apoptosis detection and EDU testing kit (Ruibo, Guangzhou, China) for cell proliferation ability detection. Furthermore, in order to evaluate mitochondrial function, the treated cells were also harvested and detected with JC-1 Kit (Keygen), mitoROS kit (AAT Bioquest, Wuhan, China), ROS Kit (Keygen), mPTP kit (BestBio, Shanghai, China) follow the manual instructions.

HK-2 cells were cultured in confocal dishes 2 d before Ang II stimulation and were then subjected to MitoTracker Red staining (Gibco, USA). After 3 × 10 min of washing with PBS, the HK-2 cells were fixed with 4% PFA at 37°C for 15 min and then washed with PBS. The HK-2 cells were treated with Triton X-100 and blocked with 10% BSA in PBS. Then, they were incubated with primary antibodies overnight at 4°C. The HK-2 cells were washed with PBS, incubated with fluorescein-labeled secondary antibodies (Invitrogen, USA) and then rinsed with PBS again. Finally, nuclei were stained with DAPI (Invitrogen, USA). Additionally, living HK-2 cells in the different treatment groups were stained with MitoSOX (Gibco, USA) and washed with HBSS. After Hoechst 33342 staining of nuclei, the HK-2 cells were immediately visualized under a confocal microscope (Olympus FV 1000 Viewer).
In addition, HK-2 cells were prepared for quantification of MMP using the JC-1 kit (KeyGEN, China). Uptake of the fluorescent dye JC-1 from the cytoplasm to the mitochondria was observed by fluorescence microscopy and was measured using Image Pro-Plus software.

Cultured in DMEM with 10% FBS, MFC were seeded and grown for 24 h. Then, the cells were treated with culture medium (set as the blank group) or Cu₂O-PEG NCs for 24 h. To explore anti-tumor mechanism, aforementioned treated cells were also analyzed with ROS (Sigma-Aldrich), Rho123 assay, mitochondrial ROS (mitoROS; AAT Bioquest, Wuhan, China), mitochondrial permeablity transition pore (MPTP) kit (BestBio, Shanghai, China), JC-1 kit (Keygen, Nanjing, China), and mitochondrial complex I–V (Solarbio, Beijing, China).

Cells treated with YCH337 for the indicated time were collected and washed with PBS. Then, the cells were stained by using a JC-1 kit (Keygen, Nanjing, China). MMP was analyzed with a FACS Calibur (BD Biosciences, Franklin Lakes, NJ).

Mitochondrial membrane potential was analyzed by JC1 kit (KeyGEN, Nanjing, China). The JC1 is a lipophilic cationic probe which can enter into the mitochondria, where it accumulates and starts forming reversible complexes called J aggregates. The J aggregates exhibit excitation and emission in the red spectrum, which is different from the green spectrum of the JC1 molecule. In this study, after exposure, 50 μL JC1 solution (final concentration was 20 nM) was added into each well of the 96-well plate. The plate was incubated for 25 min at 37 °C. Then cells were washed twice with PBS buffer and analyzed on a microplate reader (Synergy H1, BioTek, Winooski, VT, USA) at two groups of fluorescence: green (excitation wavelength: 485 nm and emission wavelength: 530 nm) and red (excitation wavelength: 530 nm and emission wavelength: 590 nm). Mitochondrial membrane potential was determined by the ratio of red to green fluorescence intensity. A decrease in the ratio indicated mitochondrial depolarization, which participates in the induction of the apoptotic signaling pathway.

After pre‐treating CT26 cells with HAP, HC, HL, or HCL for 24 h, the cells were collected, and mitochondrial functions were assessed using a MitoROS Kit (AAT Bioquest, Wuhan, China), a JC‐1 Kit (Keygen), and an mPTP Kit (BestBio, Shanghai, China), according to the manufacturers' instructions.