Alexa fluor 488 labeled annexin 5 and propidium iodide

LNCaP cells were treated with 300 nM of siRNA-DUPA or RNP₈. Un-treated cells were used as the negative control, whereas siRNA-DUPA with transfection agents was used as the positive control. The cells were processed on the third day after treatment. Cell viability was measured using the CellTiter-Blue^® (AlamarBlue) Cell Viability Assay kit (Promega, Madison, WI), following the manufacturer’s protocol. To probe the cell cycle profiles, cells were first trypsinized with TrypLE™ Express Enzyme reagent (Thermofisher, Waltham, MA) and washed twice with DPBS. After incubation with Vybrant^® DyeCycle™ Orange Stain (Thermofisher, Waltham, MA) at 37 °C for 30 min, the stained cells were analyzed on a flow cytometer using 532 nm excitation. To test apoptosis, cells were stained with Alexa Fluor 488-labeled annexin V and propidium iodide (Thermofisher, Waltham, MA) for 15 min at RT. Cell fluorescence was measured with flow cytometry.