2023-06_ICP.tab

ICP-OES
Cu, Fe, Ca, K, Mg, Mn, Na, P, S, and Zn contents were determined by ICP-OES as described in (https://doi.org/10.7554/eLife.64690). Briefly, epimastigotes were maintained for 6 days in LIT-10 % FBS-5 μM hemin medium plus different concentrations of copper or copper chelators, or in LIT-10 % FBS without hemin medium plus different concentrations of hemin or iron. For heme starved cultures, the parasites were maintained the 10 previous days at mid-log phase by periodic dilutions in LIT-10 % FBS without hemin medium before the treatments. 120 x 106 epimastigotes were collected per measurement and lyophilized. The samples were digested by boiling for 1 h in 40 % nitric acid, then diluted in ultra-pure metal-free water and analyzed by ICP-OES (Perkin Elmer, Optima 7300DV) versus acid-washed blanks. Concentrations were determined from a standard curve constructed with serial dilutions of two commercially available mixed metal standards (Optima). Blanks of nitric acid with and without ‘metal-spikes’ were analyzed to ensure reproducibility.

Reagents
The experiments were done using T. cruzi Dm28c strain. T. cruzi epimastigotes were maintained in mid-log phase by periodic dilutions in Liver Infusion Tryptose (LIT) medium (5 g/L liver infusion, 5 g/L bacto-tryptose, 68 mM NaCl, 5.3 mM KCl, 22 mM Na2HPO4, and 0.8 % (w/v) glucose, pH 7.4) supplemented with 10 % FBS and 5 μM hemin (LIT-10 % FBS-5 μM hemin medium), at 28 °C. Fetal Bovine Serum (FBS) was obtained from Internegocios S.A. (Buenos Aires, Argentina) and heat-inactivated at 56 °C for 30 min. Hemin stock solution (1 mM) was prepared in 50 % (v/v) EtOH, 0.01 N NaOH, fractionated and stored at −80 °C.
Copper (Cu) was added as Cu2+ from CuSO4 salt. Bathocuproine disulfonate (BCS, an extracellular copper chelator). Neocuproine solution (NeoCup, an intracellular copper chelator) was prepared from Neocuproine hydrochloride monohydrate.

Authors: Merli, Marcelo Luciano; Xinyu Zhu, Paul A. Cobine; Cricco, Julia Alejandra.