2022-11-08_ROS test_in triplicates_3rd.tab

Superoxide anion production in epimastigotes
Superoxide anion production was quantified as it was previously described (https://doi.org/10.1016/j.cryobiol.2013.05.005) with some modifications. Epimastigotes were maintained for 10 days in mid-log growth phase by periodic dilutions in LIT-10 % FBS medium without hemin. Then, the cells were diluted at a concentration of 5 x 106 parasites/mL and challenged to grow in LIT-10 % FBS medium without hemin plus 100 µM Cu (or no addition for controls). After 5 days, 50 x 106 cells per sample were collected, washed with PBS, resuspended in 270 µL PBS plus 30 µL of 10 mg/mL NBT (nitro-blue tetrazolium chloride), and incubated 30 min at 28 °C. After the incubation, the cells were washed with PBS and lysed with 161.6 µL DMSO. The mix was vortexed and then 138.4 µL 2 M NaOH were added. Absorbances of the samples were measured in microplate reader (Multimodal Microplate Reader, model Synergy HTX version S1LFA, BioTek Instruments) at 540 nm (the concentration of superoxide anion can be calculated using an extinction coefficient of 7.2 cm2 µmol−1).
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 broth, 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. Liver Infusion Broth BD DIFCO™ # 226920. GranuCult® prime Tryptose Broth Millipore #1.03866. Fetal Bovine Serum (FBS) was obtained from Internegocios S.A. (Buenos Aires, Argentina) and heat-inactivated at 56 °C for 30 min. Hemin (Frontier Scientific #H651-9) 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 (Supelco # 72090).

Authors: Merli, Marcelo Luciano; Cricco, Julia Alejandra.