Universal 320r centrifuge

We recorded descriptive data, including demographic variables, and, for patients, admission diagnosis, disease severity scores (Acute Physiology and Chronic Health Evaluation II score on the day of the study), serum creatinine and 90-day mortality.
Blood samples were drawn at baseline (prior to delivery of the test meal) and at 5, 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 minutes following the infusion of the test meal. Plasma samples were immediately placed in a cooled storage container and then centrifuged at 3,200 rpm for 15 minutes at 4°C (Universal 320R centrifuge; Hettich, Tuttlingen, Germany). Samples were then decanted and placed in 5-ml collection tubes and stored in a monitored −80°C specimen freezer (MDF-U74V; Sanyo Electric Co, Osaka, Japan) until analysis was performed. Serum samples were kept at room temperature for 30 to 60 minutes to allow clot formation to occur, then centrifuged as above, decanted into 5-ml aliquots and placed into a monitored −80°C specimen freezer. Serum 3-OMG concentrations were measured using liquid chromatography/mass spectroscopy. Citrulline concentrations were determined by stable isotope dilution tandem mass spectrometry (coefficient of variation, <10%). (API 4000; AB SCIEX, Framingham, MA, USA) at the SA Pathology laboratory (Women’s and Children’s Hospital, North Adelaide, SA, Australia) [24 (link)].

UHPLC–MS/MS experiments were performed in an Agilent 1290 Infinity LC (Agilent Technologies) coupled to an API 3200 triple quadrupole mass spectrometer (AB Sciex, Darmstadt, Germany) with electrospray ionization (ESI). The chromatographic separation was carried out using an Agilent Zorbax Eclipse Plus RRHD C18 column (50 × 2.1 mm, 1.8 µm). Analyst software (Version 1.6.3, AB Sciex) was used for acquisition and data analysis.
During the sample treatment procedure, a high-speed solids crusher (Hukoer, China), an evaporator system (System EVA-EC, from VLM GmbH, Bielefeld, Germany), a vortex-2 Genie (Scientific Industries, Bohemia, NY, USA), and a universal 320R centrifuge (Hettich Zentrifugen, Tuttlingen, Germany) were used.

The culture medium was removed
from a 25 cm² culture flask
on the day of the experiment, and cells were washed twice with 3 mL
of phosphate-buffered saline (PBS; GE Healthcare Life Sciences, South
Logan, UT). Subsequently, 3 mL of a plain DMEM medium (negative control)
and a DMEM medium containing TBHP (positive control) or individual
oximes were added to each flask, and cells were incubated for 1, 4,
and 24 h. The concentration of individual oxime and TBHP in the DMEM
medium corresponded to the IC₅₀ value. After incubation,
the experimental medium was removed, and cells were washed with 3
mL of PBS and harvested by scraping. The resulting suspension was
centrifuged at 220g and 21 °C for 5 min (Universal
320R centrifuge, Hettich, Tuttlingen, Germany). The supernatant was
removed and dried cell pellets were stored at −80 °C.
Each experiment was carried out in three independent replicates.

Venous blood (7 mL) was collected in EDTA tubes by trained nurses of Bama’s clinic and stored immediately on ice before transportation to the laboratories of the Institut de Recherche en Sciences de la Santé (IRSS, Bobo-Dioulasso). Vials were protected from light with aluminum foil to avoid photo-degradation of the carotenoids and retinol. Blood samples were centrifuged the same day at 3000 rpm for 10-min with a Universal 320R centrifuge (Hettich Zentrifugen, D-78532, Tuttlingen, Germany). Serum was transferred into brown 1.5 mL microcentrifuge tubes (Eppendorf, Hamburg, Germany) and stored at −20 °C until shipment to the University of Wisconsin–Madison, USA (UW) for serum retinol and carotenoid analyses. Malaria parasites were counted on thick blood smears prepared in the field from capillary blood. Hb concentration was measured on capillary blood with the 301+ Hemocue system (Angelholm, Sweden). Height to the nearest 0.1 cm and weight to the nearest 10 g were measured.

For protein extraction, we used freeze-dried biomass obtained after 14 days of cultivation. Basically, for solubilization, we left 5 mg microalgae powder suspended in 50 μL of 1 M NaOH for 10 min in a water bath at 80 °C. Then, we added 450 μL of distilled water, and the resulting solution was centrifuged for 30 min at 12,000× g (in a Universal 320 R centrifuge, Hettich, Tuttlingen, Germany), and the supernatant was moved to another tube. The above procedure was repeated twice, and the last extract was combined. The protein content was determined using two methods, Bradford and Biuret. For the Bradford method, we used bovine serum albumin (BSA) as standard in a concentration series of 0–10 µg/mL [95 (link)]. Briefly, over 200 µL of a sample, we added 50 µL of Bradford Reagent (Bradford Reagent, Sigma, Merck Group, Darmstadt, Germany), incubated for 5 min, and read the absorbance in a 96 well plate reader (CLARIOStar, BMG Labtech, Ortenberg, Germany) at a wavelength λ of 595 nm. For Biuret, the BSA standard curve was 0–10 mg/mL. Briefly, we added 200 µL of Biuret reagent to 40 µL of the sample; we incubated it in the dark at room temperature for 30 min, and then the absorbance (CLARIOStar, BMG Labtech, Ortenberg, Germany) was read at the wavelength λ = 555 nm.