Calcein am pi

The cell viability was evaluated by CCK-8 (Biosharp, China). Briefly, chondrocytes were cultured in the 96-well plates with the density of 8000 chondrocytes per well. After 24 h of culture, the medium was replaced with fresh culture medium containing PDA or PDA-Pd with concentrations ranging from 0 to 500 μg/mL for another 24 h, each concentration was repeated 3 times. Subsequently, the chondrocytes were cultured with 100 μL medium and 10μL CCK-8 for 2 h after PBS buffer washing. Finally, the chondrocyte viability was quantified by detecting the absorbance at 450 nm.
In addition, the protective effect of PDA and PDA-Pd NPs on chondrocytes against apoptosis induced by IL-1β was also evaluated by live/dead staining. In detail, chondrocytes were seeded into 6-well plates with a density of 1× 10⁵ cells per well. After 24 h of culture, the chondrocytes were attached and the medium was removed. Subsequently, after IL-1β stimulation (10 ng/mL, 24 h), chondrocytes were incubated with PDA, PDA-Pd, or PDA-Pd plus NIR irradiation (0.8 W/cm², 5 min) respectively at the same concentration of 50 μg/mL for 24 h. Then the wells were washed with PBS buffer twice. Then the chondrocytes were stained with Calcein AM/PI (Beyotime, China) for 5 min. Finally, the chondrocytes were imaged by a fluorescent microscope (Echo Revolve, China), and quantified by Image J software.

First, 2 × 10⁶ NPCs were plated onto 6-well cell plates, and after 48 h, the cells completely adhered. Then, cells were treated with different intervention factors (H₂O₂, CsA, or E6446) for 24 h. The NPCs were gently washed three times with PBS (G4202, Servicebio) and incubated with Calcein AM/PI (C2012, Beyotime) at 21 °C for 30 min. Images were obtained through fluorescence microscopy (Eclipse 80i, Nikon).

The CN-Patch cytotoxicity was evaluated using HUVECs. Briefly, log-phase HUVECs were seeded in 96-well plates (1 × 10⁴ cells per well) and cultured in 100 µL medium at 37 °C for 24 h. The prepared CN-Patch (200 µL) was soaked in PBS and incubated at 37 °C. Leach liquor was collected at 1, 12, 24, 48, 72, and 128 h to incubate the cells, and 100 µL DMEM media containing 10 µL CCK-8 (C6005, NCM, China) reagent was added to each well. HUVECs were then incubated for a further 2 h at 37 °C. Finally, the absorbance of each group was read at 450 nm using a microplate reader, where absorbance values were directly proportional to cell viability.
To exclude the direct cytotoxic effects of the CN-Patch, we further performed the Live/Dead staining on HUVECs with Calcein-AM/PI (C2015M, Beyotime, China) Double Stain Kit. Freshly prepared CN-Patch (200 µL) was soaked in PBS and incubated in an incubator at 37 °C. The leach liquor was collected to incubate the cells. Cells were collected by centrifugation and resuspended in AM/PI-buffer. The reaction solution was mixed by pipetting and incubated at 37 °C for 30 minutes. Then, the cells were visualized using an inverted fluorescence microscope. Viable cells showed uniform green fluorescence, while the dead ones were stained in red.