Ballari_et_al_Supporting_Information_02_Cell_death_experiments.pdf

Cell death experiments: cell death analysis for Trypanosoma cruzi epimastigotes.

Analysis of cell death and cell cycle arrest mechanisms on T. cruzi epimastigotes caused by treatment with proline analogues.

To analyse cell death mechanism, T. cruzi, strain CL14 epimastigotes (2.5x106 cells/mL) were cultured in LIT medium at 28 °C and treated with compounds 1, 2 and 3 at their respective IC50 or IC80 concentration. After 24, 48 and 72 h of incubation, treated cells and controls (1x106 cells/mL) were washed once with annexin buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl, pH 7.4) and resuspended in 50 µL of the same buffer. The parasites were incubated in ice (protected from light), for 15 min, in the presence of annexin-V FITC (Invitrogen, Eugene, Oregon, USA), according to the manufacturer's instructions, and 1 µg/mL propidium iodide. Then, 450 µL of annexin buffer was added, and the parasites were analysed by flow cytometry (AccuriTM C6 Plus cytometer, BD Biosciences), with 10,000 events collected and analysed using Flowjo_v10.8.1 software.

On the other hand, to explore cell cycle arrest mechanism, epimastigote forms were cultivated in LIT medium and treated with IC50 or IC80 of the compounds 1, 2 and 3 during 24, 48 and 72 h. To analyse the DNA content, the parasites were washed once in PBS and resuspended in lysis buffer (phosphate buffer Na2HPO4 7.7 mM, KH2PO4 2.3 mM, pH 7.4) and digitonin 64 μM. Then, the parasites were incubated in ice for 30 min, and propidium iodide 20 μg/mL was added. The parasites were analysed by flow cytometry (AccuriTM C6 Plus cytometer, BD Biosciences), with 10,000 events collected and analysed using Flowjo_v10.8.1 software.