2022-08-25_Effect of Cu and chelators on metaciclogenesis.tab

Quantification of metacyclogenesis:
Epimastigotes were maintained in mid-log phase by dilutions every 2-3 days in LIT-10 % FBS 5 μM hemin medium, at least for 10 days. Then, the cells were washed with PBS, diluted at a concentration of 5 x 106/mL and incubated in LIT-10 % FBS 5 μM hemin pH 6 medium for 7 days at 28 °C. The medium was supplemented with Cu, BCS or NeoCup (concentration in μM). The cells were collected, washed with PBS, fixed with 3.7 % (w/v) formaldehyde in PBS, and washed with PBS. Then, the number of epimastigotes and metacyclic trypomastigotes were counted in Neubauer chamber.
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.