Spectrophotometric microplate reader

To analyze the adhesive ability of the cells, a CCK-8 assay (Dojindo Molecular Technologies, Inc.) was conducted following siRNA transfection for 48 h. Briefly, 1×10⁴ cells resuspended in 100 µl media were seeded in each well of a 96-well plate and were incubated at 37°C for 1 h. Each group (3 wells per group) including siRNA-1, siRNA-2, siRNA-3, siRNA-NC, mock and blank, was divided into 3 groups: washed, unwashed and blank group. In the washed group, the cells were washed gently with PBS three times and 100 µl fresh media with 8 µl CCK-8 reagent was added to the wells. In the unwashed group, the cells were added with 100 µl fresh media with 8 µl CCK-8 reagent. In the blank group, CCK-8 was directly added into the wells without cells. After incubation for 2 h at 37°C, the adhered cells were determined by detecting the absorbance at 450 nm using a spectrophotometric microplate reader (BioTek Instruments, Inc.). Cell adhesion was determined using the following formula:
$\text{Cell adhesion rate}=\frac{{\text{OD}}_{\text{washed}}-{\text{OD}}_{\text{blank}}}{{\text{OD}}_{\text{unwashed}}-{\text{OD}}_{\text{blank}}}$

Cell viability was evaluated using Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan), as previously described (19 (link)). Briefly, MC3T3-E1 cells were seeded in 96-well plates at a density of 5×10⁴ cells/well, and were cultured at 37°C in a humidified atmosphere containing 5% CO₂. Once the cells reached 80% confluence, they were treated with conditioned medium prepared from α-minimum essential medium containing either different doses of Dex (0.01, 0.1, 1 and 10 µM), mangiferin (10, 20, 30, 40 and 60 µM), 1 μM Dex and different doses of mangiferin (30, 40 and 60 µM) or 1 µM Dex with or without 60 µM mangiferin treatment. At the indicated timepoints, the culture supernatant was removed, cells were washed with PBS, and 100 µl fresh medium mixed with CCK-8 solution was added to each well, followed by a further 1 h incubation at 37°C. Absorbance of the supernatant was measured at a wavelength of 450 nm using a spectrophotometric microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The cell viability assay was assessed using a CCK8 kit. All materials were sterilized using ultraviolet irradiation for 30 min. After cell attachment, media containing different concentrations of sterilized materials were added into the wells and co-cultured with the cells for 24 h. Sample free culture medium was used as the blank control. After incubation, the medium was removed and the cells were washed twice with PBS. Then, 90 μL of culture medium and 10 μL of CCK-8 solution were added into each well and incubated at 37 °C for 3 h. The absorbance of the samples was measured at 450 nm with a spectrophotometric microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
Meanwhile, the long-time influence of the as-prepared CaPs materials on the viability of MC3T3-E1 cells was also studied. The sterilized materials were dipped in cell culture medium at certain concentrations and incubated at 37 °C for 24 h. Then, the supernatant of the medium was added into the wells and co-cultured with the cells for a designated time period. The culture medium containing no extract was used as the blank control. Then, the cell viability was measured by a CCK8 kit as described as above, and the data are shown in the form of a percentage compared with the blank control from the first day.

Cell adhesion rate was analyzed by the Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). After siRNA transfection for 48 h, 3×10⁴ cells were re-suspended in culture media and 100 µl were aliquoted in each well of a 96-well plate and incubated at 37°C for 1 h. 3 wells of each group were washed gently three times with PBS, and 100 µl of fresh culture media with 8 µl of CCK-8 reagents were added. In order to analyze the total cell concentration, CCK-8 was directly added to 3 different unwashed wells. After incubation for 3 h at 37°C, concentration was determined by measuring absorbance at 450 nm using a spectrophotometric microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The cell adhesion rate was calculated as follows: