Bio sun illuminator

The antioxidant effect of BFR-EVs was evaluated by H2DCFDA staining and qRT-PCR analysis. Briefly, HDFs were seeded into 6-well plates (SPL) at a density of 1.5 × 10⁴ cells/cm² and cultured at 37 °C in a humidified atmosphere with 5% CO₂ for 24 h. HDFs were then treated with different concentrations of BFR-EVs in serum-free DMEM/F12 for 24 h. Intracellular ROS were induced by H₂O₂ treatment and UVB (315 nm) irradiation. To generate H₂O₂ induced ROS, cells were washed with PBS several times and treated with 0.5 mM H₂O₂ for 3 h. After 3 h of H₂O₂ treatment, a specific assay was performed. For UVB-induced ROS generation, cells were washed with PBS several times and 700 μL of PBS was added to each well. Cells were then irradiated with 80 mJ/cm² of UVB generated by a Bio-Sun illuminator (Vilber Lourmat, Eberhardzell, Germany). After UV irradiation, cells were maintained in serum-free DMEM/F12 for 24 h before a specific assay. Specific analysis was then performed after washing the cells several times with PBS. Intracellular ROS levels were observed by H2DCFDA staining. Cells were treated with 10 μM H2DCFDA for 30 min. Cell nuclei were stained with 2.5 μg/mL Hoechst 33342 for 20 min. Subsequently, the cells were washed three times with PBS. Intracellular ROS levels were analyzed using a fluorescence microscope and a flow cytometer (FACSymphony™ A3, BD, San Jose, CA, USA).

HDFs were plated in 96- or 24-well plates, depending on the assay, and cultured for 24 h. Then, cells were treated with 20 × 10⁸ particles/mL iPSC-Exo in serum-free DMEM/F12 for 24 h. After washing three times, 50 μL and 200 μL of PBS were added to 96- and 24-well plates, respectively. Cells were irradiated with UVB (315 nm) generated by a Bio-Sun illuminator (Vilber Lourmat, Eberhardzell, Germany). The light intensity was estimated according to the distance between the UV illuminator and the each well plate. After the UV irradiation, PBS was replaced with DMEM/F12 and the cells were further incubated for 24 h or 48 h prior to specific assays. For MTT assay, cells were divided into iPSC-Exo-untreated (Control) and treated group (iPSC-Exo). Each group was further divided into following three groups: UVB-unexposed, 40 mJ/cm² UVB-exposed, and 80 mJ/cm² UVB-exposed group.