TUNEL staining

For identifying apoptotic cells, TUNEL assay was done to find the fragmentation of DNA. For TUNEL assay, 1 × 10⁶ cells/well chondrocytes were transferred to a 96-well plate and were treated with reagents, followed by incubation at room temperature. The cells were fixed using paraformaldehyde (4%) for 20 min at 37ºC, followed by washing twice with PBS and permeabilized using triton-x 100 (0.1%) and again washed twice with PBS. The apoptotic chondrocytes were subjected to staining using the cell death detection kit for TUNEL for in vitro studies. The cell nuclei were counterstained with DAPI. After that, the slides were mounted with cover-slips followed by observation under a confocal microscope for identifying apoptosis. The percentage of apoptotic cells was evaluated as the number of fluorescein labeled cells/DAPI-stained nuclei. For calculations 100 cells from 25 different microscopic fields were selected to confirm the number of cells with apoptotic morphology.