Cck 8

Cells were seeded in 96-well plates at a density of 5 × 10³ cells/well with 100 μL medium. Cultured cells were treated with FCP, FCP/HA, HA + FCP/HA (Addition of 100 μg/L HA firstly, followed by the FCP/HA after 1 h), or cisplatin at the indicated concentrations. After 72 h, the remaining medium was discarded and another 100 μL fresh medium with 10% cell counting kit-8 (CCK-8, Solarbio) was added to the medium, and the cells were incubated for 2 - 4 h until the color of the control group turned bright yellow. The A450 intensity was measured using a Thermo Scientific Varioskan Flash multimode reader.
Cells were seeded in 96-well plates at a density of 5 × 10³ cells/well with 100 μL medium. Cultured cells were treated with FCP-Tph/HA, Tph and FCP-Tph/HA - O₂, Tph - O₂ (1% O₂ atmosphere) for 4 h, and then the cells were washed with PBS for 4 times. Fresh medium was added and irradiated by a laser with 650 nm, 200 mW/cm² for 10 min. After 20 h, the remaining medium was discarded and another 100 μL fresh medium with 10% CCK-8 (Solarbio) was added to the medium, and the cells were incubated for 2 - 4 h until the color of the control group turned bright yellow. The A450 intensity was measured using a Thermo Scientific Varioskan Flash multimode reader.

The CCK-8 method was used to detect the biocompatibility of the hydrogel leachate on MG63 cells. Briefly, the lyophilized hydrogels were immersed in a 75% ethanol solution and centrifuged to remove air bubbles. They were then repeatedly sterilized with 75% ethanol, rinsed several times with PBS, placed in a biosafety cabinet under UV light, and sterilized by ozonation. Next, 0.2 g of sterile hydrogel was incubated in 12 mL of MEM (containing NEAA), a medium containing 10% (v/v) fetal bovine serum, for 7 days at 37 °C. The leachate was filtered through a 0.22 μm nylon membrane to remove impurities. An MG63 cell suspension (10⁴ cells/mL, 200 μL) was inserted into 24-well microplates and incubated at 37 °C for 4 h, and then 300 μL of hydrogel leachate was added and incubated for 5 days at 37 °C in a 5% CO₂ incubator. The cell viability was measured using a CCK-8 reagent (CCK-8, Beijing Solarbio Science & Technology Co. Ltd., China; CCK-8-to-culture-medium volume ratio of 1/10). The absorbance values were recorded at 450 nm on days 1, 3, 5, and 7 using an enzyme marker (Epoch, Biotek, Winooski, VT, USA).

For the cytotoxicity test, RAW 264.7 cells were employed, and adherent cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum in a CO₂ incubator at 37°C. Each well of the 96-well microplates received 100 µL of cell suspension containing 10⁵ cells. After incubated for 24 h, wells were treated with 10 µL serial concentration of colistin (1 and 2 µg/mL), eugenol (31, 62, 125, 250, 500, and 1,000 µg/mL) or their combinations (1 µg/mL of colistin and 125 µg/mL of eugenol, or 2 µg/mL of colistin and 250 µg/mL of eugenol). Cells treated with 5% DMSO or left untreated served as vehicle and negative control. After 12–18 h of incubation, 10 µL of CCK-8 (Solarbio) was added.
2-(2-Methoxy-4-nitrophenyl)−3-(4-nitrophenyl)−5-(2,4-disulfophenyl)−2H-tetrazolium Sodium Salt (WST-8, key component of CCK-8 kit) can be REDOX to water-soluble yellow Formazan by NAD+, the more viable cells, the more Formazan were produced. OD_450nm was measured prior to incubating for 1 h in a CO₂ incubator. Cell viability (%) was calculated as follows: A₁−A₀/A₂−A₀ × 100%, A₁, A₂, and A₀ indicate distinct treatment groups, untreated groups, and wells exclusively with DMEM and CCK-8, respectively.