Microvette cb 300 k2e

Drops of blood were collected by fingerprick for Paracheck Pf RDT (Orchid Biomedical Systems), which detects the presence of P. falciparum histidine-rich protein 2 in blood specimens; whole-blood ribonucleic acid (RNA); thick and thin blood smears; and dried blood spots (DBS) on filter paper (903 Protein Saver; Whatman). Individuals who tested positive for asymptomatic P. falciparum infection by RDT were treated at the point-of-care using the standard regimen recommended by the Ministry of Health in Kenya. For whole-blood RNA, 200 μl of peripheral fingerprick blood was collected using capillary blood collection tubes containing Tris-ethylenediaminetetraacetic acid (EDTA; Microvette CB300 K2E; Sarstedt) and transferred immediately in cryotubes pre-filled with 400 μl Tempus solution (Applied Biosystems). Filled sample tubes were agitated vigorously per the manufacturer’s instructions and stored at − 80 °C within 24 h of collection until use.

Blood collection (300 μl) was performed at 6 weeks and 6 months of age with isoflurane-anesthetized mice (Table 2). We used a fine-walled capillary to slit slightly the retro-orbital sinus and subsequently collected blood in several tubes (Microvette CB 300 K2E) according to manufacturer’s instruction (Sarstedt AG & Co., Numbrecht, Germany). After centrifugation for 20 min at 2000 rpm (Universal 320R centrifuge, Hettich, Vlotho, Germany), the plasma was stored in aliquots at -20°C. Glucose was measured using a glucose oxidase assay (Glucose Assay Kit, abcam plc, Cambridge, UK). Leptin and insulin levels were quantified using a sandwich enzyme immunoassay (Leptin Quantikine ELISA, R&D Systems, Minneapolis, MN, USA and Rat/Mouse Insulin ELISA Kit, Merck Millipore, Darmstadt, Germany). Corticosterone was measured using a competitive immunoassay (Corticosterone EIA Kit, Lörrach, Germany). All assays were performed according to the manufacturer’s instructions and results are presented as mean±S.E.M.

In order to determine corticosterone (CORT) plasma levels, blood samples were collected at three points in time: Baseline I (Day −3), Baseline II (Day 7) and after the expression session (Day 12). For blood collection, the rats were gently restrained, a small tail vain incision was made and ~40 μl of blood was collected in ethylenediamine tetraacetic acid (EDTA)-coated microtubes (Microvette® CB 300 K2E, Sarstedt AG &Co., Nümbrecht, Germany). Samples were immediately put on ice and centrifuged at 4 °C with 3000 rpm for 10 min (Eppendorf AG, Hamburg, Germany). Plasma (~15–20 μl) was collected and stored at −80 °C until further processing. Blood samples were consistently drawn in the morning and 30 min after behavioral testing between 08:30 a.m. and 10:00 a.m. Animals were handled and habituated to the blood collecting procedure prior to the first blood collection.

Animals were housed in groups of 2–4 in Eurotype II Long cages as described earlier. All experiments started at 8:00 a.m. in the morning with a subcutaneous injection of either non-retard formulation or BUP-Depot. Mice were randomly allocated by group and sampling time point. Detailed information about weight of animals and injected doses can be found in supplement (table S2). At specific time points (0.5, 2, 5, 12, 24, 48, 72 h) animals were anesthetized using isoflurane (Attane, Lyssach b. Burgdorf, Switzerland). Anesthesia was induced with 5% Isoflurane, maintained at 3%, and the heart was punctured with a syringe. Blood was collected, transferred to EDTA coated tubes (Microvette CB 300 K2E, Sarstedt, Nürmbrecht, Germany), and centrifuged at 3′000 g, 4 °C for 10 min. 50 µL of plasma was transferred to a 96 well plate and stored at -20 °C until further analysis. Directly after blood sampling, the entire brain was carefully extracted and dissected using a scalpel or razor blade into the following tissues: cerebellum, medulla oblongata with pons and remaining brain (midbrain and forebrain). All parts were subsequently weighed and stored at -20 °C. The specific binding of BUP in brain was calculated by subtracting drug concentration in cerebellum from combined concentrations in the remaining brain.

Animals received an i.v. activity of 150 MBq ¹⁷⁷Lu-DOTATATE (or NaCl for control animals, volume corresponding to that of DOTATATE) in conjunction with either the vehicle (volume corresponding to that of one dose of rA1M, formulation as described in Section 2.5), Vamin only (Vamin 18N/l, 35 mg/200 μL, administered i.p.), rA1M only (2 × 5 mg/kg, second dose given 24 h post-injection of ¹⁷⁷Lu-DOTATATE), or a combination of Vamin and rA1M (2 × 5 mg/kg). Animals were sacrificed after 4 days. Before euthanasia, urine was collected and kept on dry ice. Blood for peripheral blood cell and reticulocyte counts was sampled from the vena saphena of non-anesthetized animals and stored in EDTA pre-coated vials (Microvette CB 300 K2E, Sarstedt). Bone marrow was harvested from the femur as described below.