Cell counting kit 8 (cck8)

Cells were seeded at 2.0×10³ cells/well in 96-well plates (Corning, NY, USA) and cultured in SSM. On each of the following 24, 48, 72, and 96 h, 10 μl of the Cell Counting Kit-8(CCK8, 7-Sea Biotech, China) were added to the medium at the same time point, followed by incubating at 37°C for 2 h. Absorbance values were determined at 450nm using ELISA reader (Model 680; Bio-Rad, USA).

Cell viability was measured using the Cell Counting Kit-8 (7Sea Biotech, Shanghai, China) according to the manufacturer’s protocol. Briefly, hDPSCs were plated at a density of 1 × 10⁴ cells per well and a volume of 100 μL in 96-well plates. The cells were starved for 24 h with serum-free medium at 37 °C in a humidified 5% CO₂ atmosphere before the test. Then the serum-free medium was replaced with different material elutes. Cells cultured in α-MEM medium containing 10% FBS served as a blank control. At each time point (24, 48, and 72 h after seeding cells), 10 μL of CCK8 assay solution was added to each well and incubated for 4 h at 37 °C. Cell number and viability were calculated by measuring absorbance at a wavelength of 450 nm on a multi-plate reader (BIO-TEK, Winooski, VT, USA). The experiments were performed in triplicate.

The prepared disks for testing with or without aging were placed in a 24-well plate with 2 mL of BHI broth. Then, 20 μL of the diluted S. mutans suspension was added to each well. After 24 h of anaerobic culture, the bacterial cells were collected by vortexing the specimen vigorously in 9.99 mL of BHI at maximum speed for 2 min using a vortex mixer (Fisher Scientific, Pittsburgh, PA, USA). The bacterial suspension of each specimen was serially diluted, inoculated on a BHI agar plate, and incubated for 1 day at 5% CO₂ and 37°C to determine the total number of CFU recovered. Five specimens of each group were subjected to CFU counts.
For the metabolic test, Cell Counting Kit-8 (7Sea Biotech, Shanghai, China) was used according to the manufacturer instruction. In brief, the bacterial suspensions obtained from the disks were prepared as mentioned above. Two-hundred microliters of the bacterial suspensions obtained from the disks were transferred to a 96-well plate, and 20 μL of CCK-8 dye solution was added to each well and incubated at 37°C in 5% CO₂ for 2 h. The absorbance at 450 nm was measured using a microplate reader (Spectra Max M5, Molecular Devices, Sunnyvale, CA, USA). Each sample was assayed in triplicate, and an average value was calculated for each sample.

The proliferation ability of the cells was measured by Cell Counting Kit-8 (CCK-8) solution (7sea biotech, Shanghai, China). Briefly, breast cancer cells were seeded on 96-well plates (Corning, USA) at a concentration of 2 × 10⁴ cells per well and incubated at 37 °C overnight. The Cell Counting Kit-8 reagents were then added to a subset of wells when cells grew for 24, 48, 72 or 96 h. After the cells were incubated for 2 h at 37 °C, we quantified the absorbance at 450 nm using a microplate reader (Bio-Rad). Each group was made in quintuplicate.

Western blotting, cell viability assays, and cell cycle distribution analysis were performed according to our previous study [27 (link)]. Antibodies against CENPI (1: 2000, catalog no: ab118796) and MCM4 (1 : 2000, catalog no: ab4459) were purchased from Abcam (Cambridge, MA, USA). An antibody against KNTC1 (1 : 200, catalog no: sc-81853) was purchased from Santa Cruz (Santa Cruz, CA, USA). An antibody against GAPDH (1 : 1000, catalog no: 5174S) was purchased from Cell Signaling Technology (Beverly, MA, USA). HRP-conjugated secondary antibodies derived from rabbits and mice were also purchased from Cell Signaling Technology [anti-rabbit IgG (1 : 5000, catalog no: 7074S) and anti-mouse IgG (1 : 5000, catalog no: 7076S)]. Cell viability was measured using a Cell Counting Kit-8 (catalog no: C008-3, 7seabiotech, Shanghai, China) following the manufacturer's instructions. Cell cycle distribution was measured via flow cytometry with PI/RNase staining buffer (catalog no: 550825, BD Biosciences).

Cells were cultured in 96‐well microplate at a density of 5 × 10³ cells/well for 24 hours. Cell viability was assessed with Cell Counting Kit‐8 (CCK‐8) (7Sea Biotech, Shanghai, China) at indicated time post‐treatment following the manufacturer's instructions. The absorbance value at 450 nm (OD450) was read with a 96‐well plate reader (DG5032, Hua Dong, Nanjing, China), to determine the cell viability.
For colony formation assay, cells were cultured in 6‐well plate at a density of 5 × 10⁴ cells/well for 24 hours. The cells were then treated with indicated concentrations of drugs and medium (control) for 24 hours. Medium was changed every 2 days. Colonies were visualized with crystal violet staining at Day 14.
For analysis of apoptosis by nuclear staining, cells were cultured in a 3.5‐cm dish, rinsed with phosphate‐buffered saline (PBS) twice and then 500 μL DMEM containing 5 μg Hoechst 33342 was added into the plates and incubated for 15 minutes in an incubator. Apoptosis was assessed through microscopic visualization of condensed chromatin and micronucleation. Apoptosis indices were calculated as the percentage of apoptotic cells among one hundred cells in a randomly selected portion. The positive rate of apoptotic cells was calculated by GD‐10.0 image analysis system.