Cell viability, apoptotic cell death, and ROS measurements

Cell viability was assessed by using a trypan blue exclusion assay. Cell suspensions were treated with 0.4% (w/v) trypan blue (GIBCO, grand island, NY, USA) at a 1:1 ratio. Viable (unstained) and dead (stained) cells were counted using a hemocytometer under the microscope. Cell viability was presented as a value relative to the number of Con Si-transfected cells.

The apoptotic cell death was examined using an FITC-annexin V apoptosis detection kit (BD Biosciences, San Diego, CA, USA) as followed the manufacturer’s protocol. Briefly, cells were incubated with 5 μL of FITC-annexin V, added 5 μL of propidium iodide (PI), and then incubated at 37 °C for 30 min in the dark chamber. The stained cells were analyzed using flow cytometry (BD Biosciences).

Levels of ROS was detected through 2’,7’-dichlorofluorescin diacetate (DCF-DA) staining method. Briefly, cells were incubated with 10 mM DCF-DA at 37 °C for 30 min in dark chamber and then analyzed using FACScan flow cytometer with excitation and emission at 490 and 530 nm, respectively [22, 36].