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Proposed procedure to demonstrate Leishmania apoptosis. To demonstrate Leishmania apoptosis, we propose to first show cell death in general by assessing the loss of viability by establishing a growth curve and the loss of plasma membrane integrity by PI staining. Second, the type of cell death should be evaluated. In case of apoptosis, at least two apoptotic markers should be highlighted, among which DNA fragmentation (by a TUNEL assay which is easier than showing a DNA ladder), cell rounding, cell shrinkage (by evaluating Forward Side Scatter (FSC) by flow cytometry), plasma membrane modifications (by Annexin V or calcein staining) and mitochondrial depolarization. Asterisks indicate markers that are not present in autophagic cells. Apoptosis kinetics should also be evaluated, showing entry of healthy cells into early apoptosis and then into late apoptosis, to distinguish apoptosis from necrosis.

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