Vacutainer

Human blood samples (4–5 ml) were collected in a K2-EDTA containing tube (Vacutainer, BD, USA) within 48 h after onset of stroke. PBMCs were subsequently isolated within 2 h as previously reported [17 (link)]. Plasma was extracted from individual participants into a procoagulant tube (Vacutainer, BD, USA) for measurement of cytokine protein levels. Mouse blood samples were obtained from the retro-orbital plexus by enucleation to rupture the ophthalmic artery. Peripheral blood samples (1–1.5 ml) were collected in a K2-EDTA containing tube (Vacutainer, BD, USA) and processed within 2 h. After centrifugation at 1500 rpm for 10 min to remove plasma, blood samples were subjected to Ficoll density centrifugation (TBD Science, Tianjin, China) to isolate PBMCs according the manufacturer’s instructions.

Saliva was collected using a swab, sized 8 × 125 mm, (SalivaBio Children’s swab, Salimetrics, PA, USA) which was placed into the buccal cavity for 90 s. The swab was thereafter transferred to a 17 × 100 mm swab storage tube (Swab storage tubes, Salimetrics, PA, USA) and centrifuged at 3000 rpm (1401g) for 15 min. The swab was subsequently removed and the saliva deposit stored at −20 °C.
Blood samples were collected from the distal cephalic vein using butterfly needles (BD Vacutainer, Becton-Dickson, Plymouth, United Kingdom) into vacuum lithium heparin tubes and clot activator tubes (BD Vacutainer, Becton-Dickson, Plymouth, United Kingdom). The tubes were centrifuged at 3300 rpm (1695 g) for 5 min. The heparinized plasma and serum obtained was transferred into cryotubes (Low Temperature Freezer Vials, VWR, Stockholm, Sweden) and stored at −20 °C.
After the clinical part of the study was completed, all samples were transported to the Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden, by a private transportation company (Temperature control, World Courier, Bangkok, Thailand), and arrived within 48 h. During transportation, the temperature was controlled, monitored and remained below −20 °C. After arrival at SLU, the samples were freeze stored at −70 °C until analysis within a maximum of 7 months after collection.

Immediately after birth, venous umbilical cord blood (mean blood volume of preterm = 2.0 mL) was filled into heparinized tubes (BD Vacutainer, Becton Dickinson GmbH, Heidelberg, Germany) by the midwives using standard blood collection sets by the same method used for peripheral blood collection. Blood was then stored at room temperature and processed within 2–3 h after birth.
For the measurement of intracellular MPO and NE amounts, blood was collected into additional EDTA tubes (BD Vacutainer, Becton Dickinson GmbH, Heidelberg, Germany).

Blood samples were collected from all calves through jugular venipuncture on d -1, T0, 0.5, 1, 2, 4, 24, 48, 72, 144, 336, 505, 672 h after castration.
Blood samples for serum amyloid-A (SAA) and haptoglobin were collected into 10-ml non-additive tubes (BD vacutainer; Becton Dickinson Co., Franklin Lakes, NJ), centrifuged for 15 min at 1.5 × g at 4°C and the serum was decanted and frozen at -80 ºC for further analysis [35 ]. The inter-assay CV for haptoglobin was 10.8%, while SAA intra-assay and inter-assay CV were 8.8% and 10.3%, respectively.
Blood samples for white blood cell count were collected into 6-ml EDTA tubes (BD vacutainer; Becton Dickinson Co., Franklin Lakes, NJ) and were measured using a HemaTrueHematology Analyzer (Heska, Lobeland, Co).

Blood was collected into lithium-heparinized tubes (BD Vacutainer^®; Becton and Dickinson, Oxford, UK) and analyzed immediately using a portable point-of-care blood gas analyzer with test cards (EPOC^® Portable analyzer system + EPOC^® BGEM test cards, Kyron Laboratories, Johannesburg, South Africa). The device measured pH, partial pressure of carbon dioxide (PCO₂), concentrations of sodium (Na⁺), potassium (K⁺), ionized calcium (iCa⁺⁺), chloride (Cl⁻), glucose, and lactate; and calculated bicarbonate (HCO₃⁻), base excess (BE), and anion gap (AG) using the Henderson-Hasselbalch equation [15 (link),16 ]. Blood samples collected into serum tubes (BD Vacutainer^®; Becton and Dickinson) were stored in a cooler box with ice packs, centrifuged within 24 h of sample collection and stored at −80 °C for 1 month until analyzed in the clinical pathology laboratory of the Faculty of Veterinary Science, University of Pretoria. Concentrations of selected serum clinical chemistry analytes were measured using a Cobas Integra 400 Plus automated biochemistry analyzer (Roche Diagnostics Ltd., Rotkreuz, Switzerland) and commercially available kits (Roche Diagnostics Ltd.). Total Magnesium (Mg), inorganic phosphate (Pi), albumin and globulin (calculated as total protein minus albumin) were measured at all time points. Urea and creatinine were only measured at TC, T0 and T6.

The experimental protocol had four groups: (1) Rats fed AL; (2) rats under DRF schedule with mealtime only from 1200 to 1400 h for 3 weeks; (3) rats fasted for 21 h, and (4) rats that were fasted 22 h and refed for 2 h (from 1200 to 1400 h). Rats were sacrificed by decapitation at 0800, 1100, 1400, 1700, 2000, 2300, 0200, and 0500 h. The controls of feeding condition, Fa and Fa-Re, were sacrificed at 1100 h and 1400 h, respectively. Immediately after sacrifice, livers were homogenized or frozen in dry ice and kept at −80°C until analysis. Two samples of blood were collected. Immediately after decapitation, for serum, blood was collected in Vacutainer^® (Becton Dickinson, Mexico City, Mexico) tubes and centrifuged at 2500g for 5 min. For plasma, blood was collected in BD Vacutainer^® (Becton Dickinson) tubes with K₂ Ethylenediaminetetraacetic acid (EDTA) and centrifuged at 1500g for 10 min.