Quantifying Intracellular ROS in C28/I2 Chondrocytes

C28/I2 chondrocytes were plated in a 96 well plate as described above. DCFH-DA probe (#D399, Thermo Fisher Scientific, USA) was employed for the determination of intracellular ROS production [15 (link)]. After incubation with 10 μM DCFH-DA for 30 min, C28/I2 chondrocytes were observed with an inverted fluorescence microscope (Olympus, Tokyo, Japan). The levels of ROS were calculated using the software Image J. Firstly, we defined the regions of interest in the fluorescent images. The integrated density value (IDV) of fluorescence was calculated. Total cells (n) in the regions were then counted. Average ROS = IDV/n.