Facsymphony a3 se cell analyzer

For FACS analysis, 1.5 x 10⁶ parasites were harvested by centrifugation, washed twice with TDB-g buffer (5 mM KCl, 80 mM NaCl, 1 mM MgSO₄, 20 mM Na₂HPO₄, 20 mM glucose, pH 7.4) and permeabilized with 50 µl saponin diluted in TDB-g (0.05% saponin for BF and 0.1% saponin for PF parasites). Cells were incubated at 37°C (BF) and 28°C (PF) for 3 min and the reaction was stopped by adding 450 µl of 10 µg/ml RNase A diluted in TDB-g. Samples were incubated at room temperature for 30 minutes and then propidium iodide was added to a final concentration of 20 µg/ml. The content of the tubes was transferred to special cytometer tubes and analyzed in a FACSymphony A3 SE Cell Analyzer using BDFacsDiva version 8 (Becton Dickinson). Percentages of each phase of the cell cycle were analyzed with FlowJo version 10.7.1.

Levels of DNA fragmentation were measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) using the In situ Cell Death Detection Kit, Fluorescein (Roche). For each sample, 1.5 x 10⁷ parasites were harvested by centrifugation and washed twice with ice cold PBS. Cells were fixed by incubating in 2% p-formaldehyde for 45 min at room temperature, washed twice with PBS, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4°C. Samples were then stained with the TUNEL reaction mix (with fluorescein-dUTP) at 37°C for 1 h. FL1-H fluorescence was measured on 10,000 events in a FACSymphony A3 SE Cell Analyzer (Becton Dickinson). Percentages of TUNEL positive cells were analyzed with BDFacsDiva version 8 (Becton Dickinson).