Diagnosis tunel

Late apoptotic cell death was determined using a Roche Diagnosis TUNEL (terminal transferase mediated dUTP-fluorescein nick end labeling) assay kit according to the manufacturer’s instructions. Briefly, HepG2 cells treated with 130 µM of Rhy for 36 h and which were washed with cold PBS. The cells were seeded after being fixed with 4% paraformaldehyde for 30 min and washed twice with PBS for 2 min. The resuspended cells were put in a permeabilization solution (0.1% Triton X-100 and 0.1% Sodium citrate) at 4 °C for 20 min, and then the cells were washed with cold PBS. The cells in 45 µL of TUNEL enzyme and TUNEL label mixture were incubated for 1 h at 37 °C in a humidified atmosphere in the dark. After being washed with PBS, cells were analyzed by a flow cytometery (FACScan Calibur, BD Biosciences, Becton–Dickinson). Acquisition and analysis of the data were performed using Cell Quest 3.0 software.

The apoptosis-inducing effect of ACHP was further evaluated using a Roche Diagnosis TUNEL (terminal transferase-mediated dUTP-fluorescein nick end labelling) assay kit as reported earlier [49 (link)]. A549 and H1299 cells were exposed to ACHP (10 µM) for indicated time points and washed with cold phosphate-buffered saline, followed by fixing using paraformaldehyde (4%) for 30 min and washing twice with phosphate-buffered saline. Thereafter, cells were treated with 0.1% Triton X-100 and 0.1% sodium citrate for permeabilization for 20 min at 4 °C, followed by washing with cold phosphate-buffered saline. The preparation was treated with a TUNEL enzyme and TUNEL label mixture for 1 h at 37 °C in the dark, followed by washing with phosphate-buffered saline and analyzed using BD Accuri C6 plus software (version 1.0.23.1).

Individual apoptotic cell death was observed using a Roche Diagnosis TUNEL (Terminal transferase mediated dUTP-fluorescein nick end labeling) assay kit in accordance with the manufacturer’s instructions. Briefly, cells treated with 10 μM of SG-1721 for 24 h were washed with cold PBS. The cells were fixed with 4% paraformaldehyde for 30 min and washed twice with PBS. The resuspended cells were put in a permeabilization solution (0.1% Triton X-100 and 0.1% Sodium citrate) for 20 min at 4 °C, and then the cells were washed with cold PBS. The cells were subsequently incubated with a TUNEL enzyme and TUNEL label mixture for 1 h at 37 °C in a humidified atmosphere in the dark. After being washed with PBS, cells were analyzed using FACScan Calibur flowcytometry (BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA).