2.6. Apoptosis

To evaluate apoptotic events, the annexin V-FITC Apoptosis Detection kit (BD Pharmingen, Heidelberg, Germany) was applied. After incubation with mistletoe extracts, tumor cells were detached, washed twice with PBS, and then reacted with 5 μL of anti-annexin V-FITC antibodies and 5 μL of DNA-intercalating dye, propidium iodide (PI), under light protection for 15 min. Samples were then subjected to an FACScalibur (BD Biosciences; 1 × 10⁴ cells per scan) and analyzed with CellQuest software version 5.1 (BD Biosciences). Possible cytotoxic effects of the mistletoe extracts were explored by staining with cell non-permeable trypan blue (Gibco/Invitrogen, Darmstadt, Germany).