The arterial blood samples were diluted in saline to a standard volume of 300 μL.
Blood samples were diluted immediately using 0.5 mL of 0.42% NaF in water at 0°C to inhibit esterase activity and thus prevent flumazenil metabolism in the samples.
After irradiation, blood samples were diluted 1 1 with RPMI 1640 medium (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen) and incubated for 6, 24 and 48 hours at 37°C in a humidified incubator with 5% CO2.
Cord blood samples were diluted using Iscove's Modified Dulbecco Medium (Sigma, SA), layered on Ficoll-Histopaque (1.077 g/ml) (Sigma, SA), centrifuged to collect the mononuclear cell layer (MNCL), then enriched for CD34+ hematopoietic stem cells (HSCs) using Dynabead magnetic separation technology according to manufacturer's instructions (Life Technologies, SA).
When the parasitemia was above 105 trypomastigotes per mL, blood samples were diluted and the number of parasites estimated with the aid of a hemacytometer.
After white blood cell count, blood samples were diluted in RPMI-1640 medium (Gibco, France) supplemented with 2 mM L-glutamate, 8 µg/ml Gentaline, and 1 mM Sodium Pyruvate in order to obtain 2×106 mononuclear cells/ml.
Blood samples are diluted in ammonium hydroxide and metals are brought to their elementary form by passing through argon plasma before being identified by mass spectrometry.
After 25 minutes of incubation at room temperature in the dark, the blood samples are diluted 1 10 in phosphate buffered saline.
In this multielement analytical technique, blood samples are diluted with ≥18 MΩ/cm water and with diluent containing 1% vol/vol tetramethylammonium hydroxide, 0.5% disodium ethylenediamine tetraacetate, 10% ethyl alcohol and 0.05% Triton X-100.
Heparinized whole-blood samples were diluted 1 4 in culture medium as described, 22 and 200 μL were incubated in duplicate with 50 μL medium containing 0 or 100 μg/mL soluble adult worm antigen (SAWA) prepared as described previously.
