S monovette serum

Blood was withdrawn directly before and 15 min after the first and directly prior the last ECT. 2 K EDTA-Gel and Serum S-Monovettes® (Sarstedt AG & Co, Nümbrecht, Germany) were used as collection tubes. The tubes were temporally stored at 4 °C up to 3 h and at room temperature (RT; 1 h, Serum S-Monovettes® only). Blood was centrifuged (2000xg, 10 min, RT: 2 K EDTA-Gel Monovettes®, 4 °C: Serum S-Monovettes®), aliquoted and kept at − 80 °C until further use. Glucose levels were assessed using an enzymatic reference method with hexokinase (Cobas 8000, module c701; Fa. Roche). Insulin levels were measured using an ECLIA (Cobas 8000, module e801; Fa. Roche) and cholesterol and triglyceride levels were analyzed using an enzymatic color test (Cobas 8000, module c701, Fa. Roche).

Venous cannulation was not successful in three hypertensive patients. In another three patients, blood could not be drawn from the cannula at later time points. We did not place a new cannula two avoid additional stressful stimuli during the experiment.
Blood samples were drawn from the venous cannula using a tube with coagulation activator (S-Monovette Serum, Sarstedt, Nümbrecht, Germany) for ACTH and a tube containing EDTA for the other blood parameters (S-Monovette EDTA, Sarstedt, Nümbrecht, Germany). The tubes were cooled directly before use and centrifuged immediately with 2,000 G and 3,570 U for 10 min (ACTH) and for 5 min (other blood parameters), respectively. Blood samples were frozen at −80 degree. Catecholamines (norepinephrine, epinephrine, dopamine) were measured using High Pressure Liquid Chromatography (CLC300, Chromsystems, Munich, Germany) and electrochemic detection (CLC100, Chromsystems, Munich, Germany). Plasma samples were analyzed using commercially available ELISA kits for ACTH (analytical sensitivity <1 pg/ml, intra-assay and inter-assay coefficients of variation <8.8%, IBL International, Hamburg, Germany) and Cortisol (analytical sensitivity <2.46 ng/ml, intraassay and inter-assay coefficients of variation <3.5%, IBL International, Hamburg, Germany).

In brief, serum samples from patients were obtained upon enrolment under standardized conditions. After an overnight fast, the blood was drawn from the cubital vein with a Safety-Multifly^® 21G (Sarstedt AG & Co.KG, Nümbrecht). Samples for analysis of fasting glucose were stored in S-Monovette^® Fluoride/EDTA (Sarstedt AG & Co.KG). Samples for the analysis of insulin and metabolome were stored in S-Monovette^® Serum (Sarstedt AG & Co.KG). All samples were centrifuged immediately after blood drawn at 500× g for 5 min. Afterwards, serum was stored at −80 °C until further analysis. HOMA-IR was calculated as follows:
Plasma samples from rats were collected after a 12-h fasting period under deep anesthesia shortly prior to euthanasia. Immediately after collection in tubes pretreated with a DPP-IV inhibitor (Merck), plasma was separated from the blood samples by centrifugation at 5 krpm for 10 min at 4 °C and stored at −80 °C.

Donor human serum (HSe) was prepared from the blood of healthy volunteers with no previous history of schistosomiasis upon written consent. Fresh blood was collected in S-Monovette^® Serum (Sarstedt, Cat. No. 01.1601), left at room temperature for 30–60 min to clot, then centrifuged twice at 2000 x g for 10 min. Serum was collected, pooled from 11 individuals and stored at -20°C until further use. Lots of commercial human serum originated from human male AB plasma, USA origin (Cat. No. H4522, Sigma-Aldrich), male AB clotted whole blood (Cat. No. H6914, Sigma-Aldrich), and from off clot pooled mixed gender, EU (Cat. No. S-106B-EU, Batch number A50618, Life Science Group).
HM supplemented with 200 U/mL penicillin and 200 μg/mL streptomycin without the addition of HSe served as a control. The medium was replenished weekly, and viability scoring was performed on day 1, 3 and 7 p.t., and then every 7 days. Developmental stages were determined by bright field microscopy using an inverted Axiovert 10 microscope (Zeiss). For each culture condition, experiments were performed at least in triplicates.
For primary screening, the culture medium contained either 20% donor or commercial (S-106B-EU) HSe. For confirmatory testing of compounds on juveniles and adult worms, commercial HSe (S-106B-EU) was supplemented in culture medium at 20%.