Dead cell apoptosis kit

NHEK-Ad cells (10⁴) were plated on 6-well cell culture plates (SPL). At 80% confluence, the cells were irradiated with 10 Gy of γ-radiation using a Gammacell^® 40 Exactor (Best Theratronics; Ottawa, Canada) at the Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute. Cells were trypsinized, washed, and stained with Dead Cell Apoptosis Kit with Annexin V-Alexa Fluor 488 and PI (BD Bioscience; San Jose, CA, USA) and incubated for 15 min at 37 °C in the dark. Cells were analyzed by MACSQuant flow cytometry (Miltenyi Biotech; Bergisch Gladbach, Germany). TUNEL assay was also used to determine the effect of DeinoPol on radiation-induced cell apoptosis according to the manufacturer’s instructions (Promega). In brief, cells were treated with cold 4% paraformaldehyde for 30 min and 0.3% Triton X-100 was added for 5 min. The TUNEL detecting solution (terminal deoxynucleotidyl transferase and fluorescence solution) was added and allowed to stand for 60 min at 37 °C. Images were acquired using an Olympus IX3 inverted fluorescence microscope (Tokyo, Japan).

Control siRNA- and Per2 siRNA-transfected AC16 cells were collected from 12-well plate after treatment with vehicle, 100 ng/ml TNFα^{35 (link)} or 100 μM of H₂O₂^{36 (link)} for 4 h and stained with Annexin-V FITC and propidium iodide (PI) using a Dead Cell Apoptosis kit (BD Sciences). Dead cells were analyzed with a LSR4 flow cytometer (BD Biosciences) and determined by Flowjo software (Annexin-V + /PI− [apoptotic cells], Annexin V-/PI + [late apoptotic and necrotic cells] and Annexin V + /PI + [end stage apoptosis and death]). The experiments were repeated three times.