2021-12-30al 1-3_Viability test_Cu and NeoCup.tab

2021-12-30al 1-3_Viability test_Cu and NeoCup Vero cells were plated on 96 multi-well plate in DMEM-2 % FBS (4 x 103 cells per well) and incubated for 24 hours. Then, the cells were washed with PBS and incubated with 200 μL of DMEM-2 % FBS supplemented with 50-500 μM Cu and 0.01-5 μM NeoCup. No addition was made in the 100 % viability control. After 72 h, the viability of cells was measured by MTT reduction colorimetric method as described (https://doi.org/10.1515/cclm.1995.33.11.813). Briefly, the cells were incubated in 180 μL DMEM-2 % FBS plus 20 μL 5 mg/mL MTT for 1 h, washed with PBS and mixed with 100 μL DMSO. Absorbance at 540 nm was measured to determine formazan production. Reagents Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Life Technologies. 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. Neocuproine solution (NeoCup, an intracellular copper chelator) was prepared from Neocuproine hydrochloride monohydrate. Authors: Merli, Marcelo Luciano; Cricco, Julia Alejandra.