Celena s digital imaging system

MC3T3-E1 cells (2000 cells/cm²) were seeded on 3% gelatin-coated coverslips and allowed to attach in cover slips. Then, FO (0–100 µg/mL) was treated in the presence or absence of 30 µM DOI. After 3 days, the cells were fixed with 4% paraformaldehyde for 10 min at 37 °C and washed three times with ice-cold phosphate buffered saline (PBS). The fixed cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature. After washing with ice-cold PBS containing 0.1% tween-20 (PBST) for 5 min, the cells were blocked with 10% donkey serum and incubated with RUNX2 antibody (1:100 in 10% donkey serum) overnight at 4 °C. The cells were stained with Alexa Fluor^® 488 secondary antibody for 2 h and counterstained with a nuclear staining die, DAPI (300 nM), for 10 min. After being washed three times with ice-cold PBST, the coverslips were mounted onto glass slides with Dako faramount aqueous mounting media and fluorescence images were captured by CELENA^® S digital imaging system (Logos Biosystems, Anyang, Gyeonggi-do, Republic of Korea).

The production of NO and ROS in zebrafish larvae was visualized using 4-amino-5-methylamino-2'7'-difluorofluorescein diacetate (DAF-FM-DA, Sigma-Aldrich Chemical Co.) and DCF-DA, respectively, 24 h after chemical treatment as previously described (Jeong et al., 2018[19 (link)]). In brief, zebrafish embryos (4 dpf) were transferred to 24-well plates and incubated with 5 µM DAF-FM-DA and 20 µM DCF-DA for 30 min and visualized using the CELENA^® S Digital Imaging System (Logos Biosystems, Anyang, Gyeonggido, Republic of Korea). Fluorescence intensities were calculated using ImageJ software (Wayne Rasband, National Institute of Health, Bethesda, MD, USA) and expressed as a percentage compared to the untreated control.

MG-63 cells (3 × 10³ cells/mL) were seeded on 3% gelatin-coated coverslips and allowed to attach on cover slips overnight. Then, the different concentrations of FO (50 µg/mL and 100 µg/mL) were treated for 7 days. DEX (100 nM) was used as the positive control. The cells were fixed with 4% paraformaldehyde for 10 min at 37 °C and washed three times with ice- PBS and permeabilized with 0.1% Triton X-100 for 10 min at room temperature followed by washing with ice-cold PBST (PBS + 0.1% tween 20) for 5 min each. The cells were blocked with 10% donkey serum and incubated with anti-RUNX2 and anti-OSX antibodies (1:100 in 10% donkey serum) overnight at 4 °C. After washing with ice-cold PBST, fluorescent dye-conjugated secondary antibody was added (Alexa Fluor^® 488 for anti-RUNX2 and Alexa Fluor^® 647 for anti-OSX), incubated for 2 h at room temperature, and washed three times with ice-cols PBST for 5 min each. Then, the cells were incubated with DAPI (300 nM) for 10 min and washed three times with ice-cold PBST for 5 min to remove excessive DAPI. The coverslips were mounted onto glass slides with Dako faramount aqueous mounting media and fluorescence images were captured by CELENA^® S digital imaging system (Logos biosystems, Anyang-si, Gyeonggi-do, Korea).