Live, apoptotic and necrotic cells were distinguished by the AO/ethidium bromide (EtBr) dual staining method 25. Lipoplexes were introduced to semi-confluent cells (8.0 ×10 4 cells/well) in 24-well plates as previously described. After 48h, cells were rinsed with PBS (200 μl/well), stained with AO/EtBr solution (100 μg/ml AO and 100 μg/ml EtBr in PBS; 10 μl/well). Excess stain was removed by rinsing with PBS (100 μl/well). Cellular changes associated with apoptosis were observed under an inverted fluorescence microscope (CKX41, Olympus, Japan) at excitation and emission wavelengths of 540 nm and 580 nm, respectively. Images were acquired at 200× magnification using Analysis Five Software (Olympus Soft Imaging Solutions, Olympus, Japan). The % live/apoptotic/early apoptotic/late apoptotic/necrotic cells were calculated as below:

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