Microtainer

Ten mice were concussed and exsanguinated within 30 seconds from being lifted out of their cages, as described for Experiment 1. The blood from each mouse was collected in five tubes; one uncoated microcentrifuge tube for serum (“serum”), one EDTA-coated tube (“EDTA”; BD Microtainer; BD Inc.) for plasma and three heparin-coated tubes (BD Microtainer; BD Inc.) for plasma. The heparin-coated tubes were either non-spiked (“Hep”), or spiked with1 μl (“Hep1”) or 10 μl (“Hep10”) of 5000 IU/ml heparin (Amgros IS, Copenhagen, Denmark), respectively. The volumes of added heparin were based on heparin concentrations (5–50 IU/ml) often used in flushing solutions for catheters [28 (link)-30 (link)]. As the levels of five cytokines in Experiment 1 were below the standard curve and due to general low physiological concentrations of the cytokines, the samples in Experiment 2 were spiked with 10 μl of a cytokine standard (Bio-Plex Pro Mouse Cytokine Standard, Lot #5025850, Bio-Rad Laboratories, Copenhagen, Denmark), containing a known concentration of the 23 cytokines that were to be measured. The standard was added undiluted to each sample; resulting in a total volume of 130 μl. Samples were spiked with heparin and cytokine standards immediately prior to analysis.

Upon sacrifice on the seventh day, blood samples were collected from the retroorbital plexus puncture by using EDTA (Ethylenediamine Tetra-Acetic Acid) and BD Microtainer®. Approximately, blood was collected in each tube per mouse in a microcontainer and used for hematological and biochemical analyzer. Briefly, EDTA microcontainer blood was used for white blood cell (WBC) analysis by mixing thoroughly for 3-5 minutes with an automatic rolling mixer and was counted by hematology analyzer (Hemavet® HV950 FS; Drew Scientific, Erba Diagnostics, Inc., Dallas, Texas, USA). BD Microtainer® blood samples from each mouse were further centrifuged at700 − 1,000 × gfor 10 min at 4°C to get the serum and were kept at -80°C until analysis.

Blood was collected by cardiac puncture under deep anesthesia (surgical plane) using a 1-mL syringe fitted with a 23-G needle. Blood samples were split into two fractions. The first fraction was collected in K₂EDTA-coated tubes (BD Microtainer—365974) for complete blood count (CBC). CBC was performed by the Unit for Laboratory Animal Medicine—In Vivo Animal Core (ULAM-IVAC) using a Hemavet 950FS (Drew Scientific). The other fractions were collected in serum separator tubes (BD Microtainer—365967). Blood was allowed to clot for 30 min at room temperature prior to centrifugation for 5 min at 10 000 g. Samples were stored at −80 °C prior to EPO, Opg, and Rankl quantification by ELISA according to the manufacturer’s instructions (RnD Systems—MEP00B, MOP00, and MTR00).^{8 (link),12 (link)}

Mice were anesthetized by pentobarbital sodium (Entobar inj., Hanlim Pharm. Co., Ltd., Korea) injection (i.p.). Blood samples were taken and the animals were sacrificed by exsanguination from the aorta. A complete gross observation was performed on all terminated animals. The blood samples were collected in a microtainer (Becton, Dickinson and Company, NJ, USA) containing K₂-EDTA. Plasma samples were collected after centrifugation at 10000 rpm and stored at -80°C until they were further assayed. The dorsal skin and one ear of each mouse were removed and fixed in 10% (v/v) natural buffered formalin for 24 hr. The tissues were embedded in paraffin and then sectioned at 4 μm thickness. The tissue sections were then stained with hematoxylin & eosin (H&E) or toluidine blue to estimate epidermal inflammation (hypertrophy and infiltration by inflammatory cells) and mast cell counts, respectively. The dermal mast cell content was quantified by counting the numbers of toluidine-blue positive cells in randomly selected high power fields for each specimen.

Finger stick blood was first placed on a clean glass slide and prepared for thin and thick blood film staining by Giemsa in the standard manner. Certified malaria microscopists read at least 100 visual fields of oil immersion (1000X) magnification of the stained thick film prior to considering a slide negative. Positive slides were reported according to species observed in the microscopic examination from thin blood film. Blood from finger stick of volunteers was placed in EDTA micro-tubes (Becton-Dickinson Microtainer), placed in a cool dark container, and within 8hr was quantitatively assayed for G6PD activity using a commercially available kit (Trinity Biotech, Ireland; Cat. No. 345-B) with deficient (Cat. No. G5888), intermediate (Cat. No. G5029) and normal (Cat. No. G6888) G6PD controls. The reaction was read at 340nm wavelength using a Biochrom (UK) WPA Biowave II UV/Vis spectrophotometer. G6PD activity was calculated from that optical density reading as instructed by the kit manufacturer. Ten microliters of EDTA blood from the microtube was put into a micro-cuvette supplied by the manufacturer of the HemoCue system (HemoCue AB, Sweden) and immediately read in the instrument (Hb201+) of that system for hemoglobin measurement prior to the G6PD quantitative assay.