Ra fls

The RA-FLS migration assay was conducted using a transwell polycarbonate membrane (6.5 mm insert, 8.0 μm pore size). The inserted membranes were filled with 200 μL of RA-FLSs (5 × 10⁴ cells; Cell Applications, Inc., San Diego, CA, USA) containing 1% FBS, reagents, and 100 ng/mL Dex. The bottom plate was treated with 10 ng/mL TNF-α, 10 ng/mL EGF, and 50 ng/mL IL-6 dissolved in 500 μL of DMEM, and incubated for 6 h. DMEM on the bottom plate was removed, and chilled methanol was added carefully. The inserted membranes were then soaked for 1 min at −20 °C. The chilled methanol was removed, and the inserted membranes were washed three times with phosphate-buffered saline (PBS). The DAPI solution was mixed with distilled water and loaded onto the bottom plate. The plates were then incubated for 30 min in the dark. The medium was carefully removed from the insert. Distilled water (DW) was added to the bottom plate at room temperature, and DAPI fluorescence of the inserted membrane was measured at 405 nm using an LSM 700 Zeiss confocal microscope (Carl Zeiss, Jena, Germany). RA-FLS migration was determined based on the number of nuclei stained with DAPI on the transwell membrane.

The normal human FLSs and rheumatoid arthritis (RA) FLSs were purchased from Cell Applications (San Diego, CA, U.S.A.). The cells were cultured in DMEM containing 10% FBS and stored in a humidified incubator with 5% CO₂ at 37°C.

An LED-based device (width × length × height: 4.9 × 4.9 × 1.3 cm, Color Seven Co., Seoul, Korea) was used for PBM with the following parameters: wavelength, 610 nm [full width at half maximum, 24 nm (orange color)]; power intensity, 5 and 10 mW/cm²; energy density, 12 J/cm²; surface area of electrodes, 1.6 cm; spot size of electrodes, 4 mm diameter.
Fibroblast-like synoviocytes from RA patients (RA-FLSs) were purchased from Cell Applications (San Diego, CA, USA) and cultured in synoviocyte Growth Medium (Cell Applications) at 37°C with 5% CO₂. The medium was replaced every two days. For PBM treatment, RA-FLSs were seeded in triplicate, at a density of 5 × 10⁴ cells/well in 24-well plates (SPL Life Sciences Co. Ltd., Pocheon, Korea). After 18 h, the culture medium was changed to serum-free medium, and the cells were exposed to 10 ng/mL of tumor necrosis factor-α (TNF-α, Quimigen, Madrid, Spain) and/or PBM at 610 nm wavelength for 20 min in the dark; the corresponding power intensity were 5 and 10 mW/cm², respectively. Control cells were treated in the same manner, except for the light exposure. Subsequently, the cells were collected and tested for viability, proliferation, migration, invasion, and Western blot analysis.