Live dead cell staining kit

For all the experiments on cell viability and metabolic activity assays, C57BL/6 bone marrow-derived MSCs were used, which were purchased from Cyagen Biosciences (MUBMX-90011, Santa Clara, United States). Live-Dead Cell Staining Kit (Thermo Fisher Scientific, Waltham, MA, United States) was used to assay the MSCs viability and CCK-8 kit (Cell Counting Kit-8; Dojindo Laboratories, Kumamoto, Japan) was used to test the metabolic activity of MSCs growing on the hydrogels. Using the same experimental conditions, we also carried out the cytotoxicity test of LAP.

The live/dead cell staining kit (Thermo Fisher Scientific Inc., Waltham, MA) was utilized to assess the cell viability of L929 cells incubated with PEG/ZIF-8, HF, and PEG/ZIF-8@HF. First, L929 cells were introduced into a 96-well plate with the cell concentration of 1.0 × 10⁴ cells/well and cultured for 24 h. Then, PEG/ZIF-8, HF, and PEG/ZIF-8@HF with HF content of 7.5 μM were cultured with the cells for 24 h, and this concentration was also used in subsequent experiments. Then, the culture media were discarded and the cells were rinsed with PBS. Subsequently, 60 μL of culture medium containing propidium iodide (5 μM) and calcium-AM (2 μM) was introduced into each well and incubated for 0.5 h. At last, the cells were observed with a fluorescence microscope (Olympus, Tokyo, Japan).

Human epidermal keratinocytes (HaCaT; CLS GmbH, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GenDEPOT, Katy, TX, United States) containing 10% fetal bovine serum (FBS; GenDEPOT), 100 U penicillin, and 100 μg/ml streptomycin (GenDEPOT). The cells were then incubated in a humidified incubator with 5% carbon dioxide (CO₂)/95% air. Cells (1 × 10⁴ cells) were plated in a 96-well plate (SPL Life Sciences, Pocheon, Korea) and stabilized for 24 h. The dried EJ-AuNPs or EJ was diluted with serum-free medium (SFM) at concentrations of 25, 50, and 100 μg/ml. After the cells were washed twice with phosphate-buffered saline (PBS), diluted EJ-AuNPs solution was added to the cells and incubated for a further 24 h. To compare the cytotoxic effect of EJ-AuNPs, only SFM and commercial dexamethasone (20 μg/ml)-containing SFM were added to the cells. The cytotoxic effects of EJ-AuNPs against HaCaT cells were measured using a conventional 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO, United States) and a live/dead cell staining kit (Thermo Fisher Scientific, Cambridge, MA, United States), according to the manufacturer’s instructions.