Detecting Apoptosis-Induced DNA Fragmentation

Fragmented DNA was detected in situ by the terminal deoxynucleotide transferase (TdT)-mediated binding of 3′OH ends of DNA fragments generated in response to apoptotic signals, using a commercially available kit (TdT FragEL DNA Detection Kit) from Millipore Sigma (Burlington, MA, USA) as described earlier [16 (link),17 (link),18 (link)]. Coverslips containing A549 lung adenocarcinoma cells cultured to 70–80% confluence were fixed with chilled methanol (Fisher Scientific, Waltham, MA, USA) for an hour, followed by two brief rinses with sterile PBS. Cover slips were treated with 20 mg/mL proteinase K for 5 min at room temperature and washed in PBS before TdT staining. Samples were equilibrated for 30 min in 1xTdT buffer and washed with PBS prior to terminal deoxynucleotidyl transferase and DAPI (1:10,000, Millipore Sigma, Burlington, MA, USA) staining. After mounting coverslips and drying overnight, slides were visualized under the Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera.