Facscan laser flow cytometer

Caco-2 cells in logarithmic phase at were treated with test samples for indicated time. Then they were harvested, washed and resuspended with PBS. Apoptotic cells were determined with an FITC Annexin V Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer’s protocol. Briefly, the cells were washed and subsequently incubated for 15 min at room temperature in the dark in 100 ul of 1 × binding buffer containing 5ul of Annexin V-FITC and 5 ul of PI. Afterward, apoptosis was analyzed by FACScan laser flow cytometer (Guava easycyteHT, Millipore, CA).

The production of cellular ROS, mainly H₂O₂, was detected using the DCFH-DA fluorescence assay. Briefly, cells were seeded in 24-well plates at the density of 70 ~ 80% confluence per well for overnight incubation. After treatment with appropriate concentrations of test samples, cells were harvested, placed into 1.5 mL round-bottom polystyrene tubes, and washed with PBS twice. Subsequently, the cells were centrifuged for 5 min at 400 × g at room temperature, and the supernate was discarded. The cells were resuspended in 500 μL ROS detection solution, stained in the dark at 37°C for 30 min, and analyzed by FACScan laser flow cytometer (Guava easycyteHT, Millipore, CA).

HK-2 cells in logarithmic phase in a 6 well tissue culture plate were treated with test samples for indicated time. Then they were harvested, washed and resuspended with PBS. Apoptotic cells were determined with an FITC Annexin V Apoptosis Detection Kit (Beyotime, China) according to the manufacturer's protocol. Briefly, cells were washed and incubated for 15 min at room temperature in the dark in 100 μL of 1 × binding buffer containing 5 μL of Annexin V-FITC and 5 μL of PI. Apoptosis was analyzed by FAC Scan laser flow cytometer (Guava easycyte HT, Millipore, CA).