Actin tracker green 488

Chondrocytes were seeded in confocal dishes (1 × 10⁵ cells), and then incubated with Cy5.5-Cu_1.5MH or Cy5.5-Cu₆MH (20 μg/ml) in the dark for 24 h. After incubation, chondrocytes were washed with PBS 3 times and then fixed with paraformaldehyde (4% PFA, Biosharp, China) for 10 min. Later, the cytoskeleton was stained with the actin-tracker green-488 (Beyotime Biotechnology, China) following the protocols, and the nuclei was stained with 4, 6-diamidino-2-phenyindole dilactate (DAPI, Beyotime Biotechnology, China) for 10 min before PBS washing 3 times. Finally, the images were captured with a confocal scanning microscope (Leica, Germany), and the corresponding fluorescence intensity was quantified by ImageJ. Specifically, Cy5.5-Cu_1.5MH and Cy5.5-Cu₆MH were prepared by the following procedures. In detail, 50 mg of Cu_1.5MH or Cu₆MH was dispersed in DMSO followed by the addition of 50 mg/ml Cy5.5 N-hydroxysuccinimide ester (Cy5.5 NHS ester, Lumiprobe, USA) overnight. The final product was collected by vacuum drying after methanol washing. Besides, chondrocytes were incubated with Cu₆MH (20 μg/ml) for 12 h, and chondrocytes were also embedded in paraffin and cut into slice for observation by TEM.

To
monitor the cell morphology (cytoskeleton), the cells were fixed with
4% paraformaldehyde for 30 min. After washing three times with PBS,
the cells were blocked by 5% BSA for 1 h at 37 °C. Subsequently,
the cells were incubated with Actin-Tracker Green-488 (1:100; Cat#
C2201S, Beyotime, Shanghai, China) at room temperature for 1 h in
a dark environment. The Actin-Tracker Green-488 probe is a phalloidin
labeled by the fluorescent dye Alexandra Fluor 488. The nuclei were
counterstained with a DAPI solution (Cat# C1005, Beyotime, Shanghai,
China). The stained cells were imaged by using a confocal fluorescence
microscope.