Hypoxanthine

Complete medium (CM) for P. falciparum asexual and gametocyte culture was prepared by mixing 500 mL of sterile liquid RPMI medium (RPMI-1640-R5886, Sigma, UK), hypoxanthine (Sigma-Aldrich, UK) to final concentration of 0.05 g/L, l-glutamine powder (Sigma-Aldrich, UK) to final concentration of 0.3 mg/L and sterile human serum (10% of final volume). CM was aliquoted in 50 mL tubes and stored at 4 °C to be used within a week. Each batch of human serum was tested for supporting the development of healthy gametocytes as determined by optimum exflagellation activity.
Ookinete medium (OM) was prepared by dissolving 500 g of RPMI powder with l-glutamine (Sigma-Aldrich, UK), 2 g NaHCO₃ and 50 mg of hypoxanthine in 1 L distilled H₂O (dH₂O), and stored in sterile 2 L conical flask at 4 °C.
A stock of 100 mM xanthurenic acid (XA) was prepared by adding 205.17 mg XA obtained from Sigma-Aldrich (UK) to 10 mL dH₂O and 1 mL is added to 1 L ookinete medium. The solution was filtered through 0.2 μm filter units after the pH adjusted to 7.4. It was aliquoted and stored at 4 °C for use within 6 months.
A 10% fructose solution for mosquito sugar feeding was prepared by dissolving 10 g fructose (Sigma, UK) in 1 L distilled water, filtered through a 0.22 μm filter and kept at 4 °C.

AuCl₄H₂O, sodium citrate, hypoxanthine, hydroquinone, 10 nm gold colloid suspensions (6 × 10¹² /mL) and hypoxanthine were purchased from Sigma Aldrich (Ireland). Sterile filtered human serum from normal mixed pool (off the clot) was purchased from TCS Biosciences (Ireland). Uric acid was purchased from FUJIFILM Wako Pure Chemical Corporation.
Five saliva and five urine samples were collected from volunteers from the Technological University, Dublin. The participants were informed that the study is scientifically relevant and not invasive, and their actions would contribute to developing a cheap, fast and simple method, which will improve population health. Verbal informed consent was provided by the participants, who agreed to contribute anonymously.
Commercially available centrifugal filtering devices, Amicon Ultra-0.5 mL (Millipore—Merck, Darmstadt, Germany), with cut-off points at 3 kDa, were employed in this study, as examples.

Prior to injection, blood was stored at 4°C and washed 2x with RPMI 1640, 25 mM HEPES (Sigma-Aldrich, St. Louis, MO) containing 7x10^-3 mM hypoxanthine (Sigma-Aldrich, St. Louis, MO) at RT. The buffy coat was removed by aspiration with erythrocytes resuspended at 50% hematocrit in RPMI 1640, 25% decomplemented human serum (Sigma-Aldrich, St. Louis, MO), and 3.1 mM hypoxanthine. huRBC suspension was warmed at 37°C for 20 min prior to intraperitoneal (i.p.) injection. Mice were i.p injected with 1 ml of huRBCs containing 70% O⁺ human erythrocytes in RPMI 1640 (25 mM HEPES, 2 mM L-glutamine) supplemented with 50 μM hypoxanthine plus 10% human serum. Engraftment continued daily using 1 ml huRBCs throughout the experiment, in order to reach the minimum desired percentage of huRBCs ≥ 70% (measured by flow cytometry as previously described [33 (link)]) by 7 days post mosquito bite challenge.