Cck 8

Murine L929 cells and EaHy926 were used to assess cytotoxicity of PELA hydrogel and its extracts by Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), supplemented with 50 U/mL penicillin and 50 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. PLEL hydrogel was firstly extracted using DMEM with 10% FBS for 24 h, after which sequential dilutions of the stock solution were carried out to obtain leachates in series concentrations. Cells were detached using 0.25% trypsin–EDTA (Invitrogen, Rockford, IL, USA) and distributed in a 96-well plate at a density of 3 × 10⁴ cells/well and incubated for 24 h. Fresh medium with different concentrations of PLEL hydrogels or their leachates were added and incubated for another 48 h. Subsequently, CCK-8 was applied according to manufacturer’s instructions. The cytotoxicity was expressed as the relative viability (%), with no hydrogel or hydrogel leachate in culture media as 100%.

Each experimental group digested and resuspended in full culture media. Cell proliferation was measured at 1d, 2d, 3d, and 4d using the CCK-8 (Invitrogen, USA). In 450 nm, optical densities were measured using an enzyme marker (Molecular Devices, Rockford, IL).

Cell viability was analyzed using the cell counting kit-8 (CCK8; Invitrogen). Briefly, 1 × 10⁴ SKOV3 cells were seeded into a 96-well plate and incubated overnight at the previously described conditions. Then, the medium was discarded and the cells were washed with PBS three times before adding DMEM (90 µl) and CCK8 (10 µl). After incubation for 1.5 h at 37 °C, the optical density (OD) was measured with a microplate reader at 450 nm.

3T3 cells were used to analyse the cytocompatibility and cell proliferation of the hydrogel scaffolds using CCK8 (Invitrogen). The hydrogel samples were set as experimental groups and were immersed in DI water to exclude the uncrosslinked agents.
All groups were added to each well and co-cultured with the cells in triplicate. Each hydrogel group was added to a 24-well plate (n = 3). Samples weighing 20 μg and a 10 μL cell suspension with a concentration of 1 × 10⁶ cells/mL were added to the top of the hydrogels and allowed to settle for 2 h before the addition of 1 mL full cell culture media to the well. Cells with a concentration of 10,000 cells per well were used as the control group and allowed to attach overnight. The cells were incubated at 37 °C and the test following standard protocol was performed at 24, 48, and 72 h after coculture.
To visualise the cell status, a live/dead assay was used to confirm cell living status as the protocol described at 48 h, with calcein AM staining used for live cells (green colour) and ethidium homodimer-1 for dead cells (red colour). Images were taken using an inverted fluorescence microscope (Olympus IX81, Beijing, China).

Cell proliferation was determined using cell counting kit 8 (CCK8; Dojindo or PrestoBlue; Invitrogen) according to the manufacturer’s protocol. Briefly, 5 × 10⁴ of each stable cell lines were plated in 96-well plates in DMEM medium supplemented with 1% heat-inactivated fetal bovine serum (FBS). 10 µl of substract solution were added daily to each well, followed by incubation for 4 h. The cell viability in each well was determined by reading the optical density at 450 nm for CCK8 or by measuring the fluorescence intensity (RFU) with PrestoBlue at 560/590 nm on a TECAN SPARK 10 M (TECAN).

The proliferation rates of ASMCs was determined using a cell counting kit-8 (CCK-8; Invitrogen, USA). Cells in each group were inoculated in 96-well plates with 1 × 10⁴ cells per well and three repeated wells for each group. Each group was incubated for 0, 24, 48 and 72 h at 37 °C. Then, 10 μl CCK-8 solution was added to each well, and incubation terminated after 2 h. The optical density (OD) value of each well was measured at 450 nm with a microplate reader (Hanbio Biotechnology Co.). The corresponding OD value represents cell proliferation.