2024-06-20_08_Intracelular heme_Cu 500uM treatment.7z

The file “2024-06-20_SAMPLES.xlsx” shows the samples list for Heme content determination. The spectrums are recorded in separate files named as " PVC WAVE-SCAN 2024-06-20 15..." Method: Heme content determined by pyridine method Intracellular heme content determined by pyridine method in epimastigotes grown in LIT-10 % FBS-5 μM hemin medium plus 500 μM Cu or without addition (control) for 6 days. The heme content was measured by the alkaline pyridine method described in (10.1016/0003-2697(87)90643-9). We made modifications to perform the assay with epimastigotes (https://doi.org/10.1371/journal.pntd.0004359). Briefly, 50-200 x 106 epimastigotes were collected, washed twice with PBS, and suspended in 500 μL PBS. The sample was combined with a volume of 500 μL containing 0.2 N NaOH and 40 % (v/v) pyridine. Reduced spectra were recorded by addition of sodium dithionite. Heme concentration should be calculated from the differential -reduced minus oxidized- spectrum. Molar extinction coefficient at 557 nm: 23.98 mM−1 cm−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, 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. Authors: Merli, Marcelo Luciano; Cricco, Julia Alejandra.