To analyze (R-X-R) 4 -PMO conjugates cell internalization, exponentially growing HeLa pLuc705 cells (1.75 Â 10 5 cells seeded and grown overnight in 24-well plates) were incubated in OptiMEM with FAM-labeled (R-X-R) 4 -PMO. Cells were then washed twice with PBS, detached by incubating for 5 min at 378C with 0.5 mg/ml trypsin per 0.35 mM, EDTA.4Na and washed by centrifugation (5 min, 900g) in ice-cold PBS containing 5% FBS. The resulting cell pellet was resuspended in ice-cold PBS containing 0.5% FBS and 0.05 mg/ml propidium iodide (PI) (Molecular Probes, Eugene, OR, USA). Fluorescence analysis was performed with a BD FacsCanto flow cytometer (BD Biosciences, San Jose, CA, USA). Cells stained with PI were excluded from further analysis. A minimum of 20 000 events per sample was analyzed.