2022-09-30_Oxygen consumption with Cu_BCS_NeoCup_day3.tab

2022-09-30_Oxygen consumption with Cu_BCS_NeoCup_day3
Oxygen consumption was measured in epimastigotes grown in LIT-10 % FBS-5 μM hemin medium plus copper (Cu), BCS, NeoCup or no addition (control), for 3 days. Concentration in μM.
The oxygen consumption rates from epimastigotes were quantified from the linear response, as previously described*, using a Clark electrode connected to a 5300 Biological Oxygen Monitor (Yellow Springs Instrument Co.). Epimastigotes were collected, washed with PBS, suspended in TSB-EGTA (125 mM sucrose, 65 mM KCl, 1 mM MgCl2, 2.5 mM KH2PO4, 10 mM Tris-HCl pH 7.5, and 333 μM EGTA) at a ratio of 5 × 105 cells/μL and kept in ice. Before measurements, 5 mM Succinate and 50 μM digitonin were added to each sample. For oxygen consumption, 50-150 μL of permeabilized parasites were added to 1.9 mL of TSB-EGTA plus 5 mM succinate at 28 °C.
* Vercesi AE, Bernardes CF, Hoffmann ME, Gadelha FR, Docampo R. Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ. J Biol Chem. 1991 Aug 5;266(22):14431-4. PMID: 1860850.

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; Cricco, Julia Alejandra.