2022-01-17_Effect of Cu and chelators in the infection_3rd.tab

Infection assay:
Vero cells (ATCC CCL-81) were seeded on 12 mm round coverslips in 24-well culture plates, at a density of 1.58 x 103 cells/cm2, and incubated in DMEM- 2 % FBS. After 24 h, they were infected with cell-derived trypomastigotes (MOI of 10) for 16 h in DMEM-2 % FBS supplemented with 50-150 μM Cu, 50-500 μM BCS, or 0.005-0.02 μM NeoCup. Then, non-internalized trypomastigotes were washed twice with PBS and the medium was renewed. 72 h post infection, the monolayers of Vero cells were washed with PBS, fixed with methanol for 15 min, washed with stabilized water, and stained with Giemsa’s reagent for 30 min. Finally, they were washed with stabilized water, air dried, and mounted with Canada balsam. 200-300 cells per slide were analyzed by light microscopy to determine the number de amastigotes per cell. 0 indicates a cell without amastigotes.
Reagents:
Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Thermo Fisher Scientific (#12100046). Fetal Bovine Serum (FBS) was obtained from Internegocios S.A. (Buenos Aires, Argentina) and heat-inactivated at 56 °C for 30 min.
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 (Supelco # 72090).
Authors: Merli, Marcelo Luciano; Cricco, Julia Alejandra.