Evaluation of Phosphatidylserine (PS) Externalization

Externalization of PS is a marker of apoptosis. After an apoptotic stimulus, PS normally found in the inner leaflet of the plasma membrane, translocates to the outer leaflet of the membrane (Sen et al., 2007). PS is a phospholipid present in the plasma membrane of Leishmania parasites (Tripathi and Gupta, 2003) and the externalization of PS residues in apoptotic cells can be detected by annexin V staining (Taruvai Kalyana Kumar et al., 2016). After treatment with PF-429242 (3 and 6 μM), the cell concentration was adjusted to 1 × 10⁷ cells/mL in 200 μL calcium buffer, 1 μl annexin V-FITC (Invitrogen) was added to each of the samples then incubated for 30 min. The samples were read in a spectrofluorimeter at 485/528 nm (excitation/emission). Treatment with miltefosine (8 μM) was used as a positive control and promastigotes maintained in culture medium were used as a negative control. Three independent experiments were done. In parallel, PI staining was carried out to control for the integrity of the plasma membrane of the cells and to avoid staining of PS on the inner leaflet of the plasma membrane.