Becton dickinson facsort flow cytometer

The assessment of ROS production was performed using the fluorescent dye, DCFH-DA, by confocal microscopy and flow cytometry, respectively. Briefly, HT29 cells transfected with siRNAs were incubated with TNFα for 48 h. Then, the cells were washed with PBS and incubated with 10 μM DCFH-DA at 37°C for 30 min. The ROS generation was checked by DCF fluorescence, and the fluorescent images were collected by a single rapid scan using identical parameters for all samples. To measure the ROS concentration by flow cytometry, cells were loaded with 10 μM DCFH-DA for 30 min, trypsinized, and washed with cold PBS two times followed by fluorescence-activated cell sorting analysis using the Becton-Dickinson FACSort flow cytometer (BD Biosciences, NJ, USA).