Trypan blue exclusion technique

Tissue samples for PHH isolation were obtained from macroscopically tumor-free areas of resected livers (Table 1; D1–4 and DH2–10). PHHs were isolated as described previously [19 (link),20 (link),21 (link)] by a two-step EGTA/collagenase perfusion technique. Cells were pooled, washed with phosphate-buffered saline (PBS, Gibco, Paisley, UK) and resuspended in PHH culture medium (William’s Medium E, GlutaMAX™ (WME, Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Merck Biochrom, Berlin, Germany), 100 U/100 μM penicillin/streptomycin, 1% nonessential amino acids (MEM NEAA 100×), 15 mM HEPES, 1 mM sodium pyruvate (all provided by Gibco, Paisley, UK), 1 μg/mL dexamethasone (Fortecortin^®, Merck, Darmstadt, Germany) and 80 IU/l human insulin (Lilly Deutschland GmbH, Bad Homburg, Germany/Sanofi-Aventis, Frankfurt am Main, Germany). Cell count and viability were determined using a Neubauer chamber with the trypan blue exclusion technique (Sigma-Aldrich, St. Louis, MO, USA).

Purified parasites (6 × 10⁶ cells) were exposed to increasing concentrations of DHEA (1, 10, 20, 50, 80, 100, and 200 μM), of sulfadiazine–pyrimethamine (80, 200, 400, 600, and 800 μM), and to the combined treatment DHEA/S-P (80/80, 200/200, 10/600 and 10/800 μM). All drugs were diluted in PBS 1X to the final concentrations tested. The exposition was held for 30 min or 120 min at room temperature (RT) with gentle agitation. Additionally, we used a control of ethanol (Sigma), as the primary diluent of DHEA, at a final concentration of 2 µM. The ratio between live and death tachyzoites was measured by the trypan blue exclusion technique (cat. 15250–061, Gibco, Thermo Fisher, NY, USA). Three hundred parasites were counted under an optical microscope (AxioObserve A.1 Microscope, Carl Zeiss Mexico). This assay was performed in triplicate in at least three independent assays.