Vacuette k3edta tube

Whole mice trunk blood (postprandial) for CBC and OFT was placed into VACUETTE K3-EDTA tubes (Greiner Bio-One, Kremsmünster, Austria), and stored at +4 °C or room temperature before the experiment (samples were analyzed within 1–3 h after blood collection). After hematological measurement, red blood cells (RBC) of each sample were isolated for the osmotic fragility test.
Serum for biochemical screening (postprandial): rats were shortly anesthetized with isoflurane and killed by decapitation; trunk blood was collected into VACUETTE blood collection tubes for serum (Greiner Bio-One, Kremsmünster, Austria), incubated in a vertical position for 15 min, and then kept at +4 °C until centrifugation. Samples with coagulated blood were centrifuged at 1500 rpm for 15 min at +4 °C. Serum was transferred into dry clean tubes and stored until analysis at −20 °C for no more than 5 days.

Whole blood (postprandial) for hematological parameters and osmotic fragility test (OFT): rats were shortly anesthetized with isoflurane and killed by decapitation. Trunk blood was placed into VACUETTE K3-EDTA tubes (Greiner Bio-One, Kremsmünster, Austria), and stored at +4 °C or room temperature before the experiment (samples were analyzed within 1–3 h after blood collection). After hematological measurement, red blood cells (RBC) of each sample were isolated for osmotic fragility test [20 (link)].
Serum for biochemical screening (postprandial): rats were shortly anesthetized with isoflurane and killed by decapitation, trunk blood was collected into VACUETTE blood collection tubes for serum (Greiner Bio-One, Austria), incubated in a vertical position for 15 min, and then kept at +4 °C until centrifugation. Samples with coagulated blood were centrifuged at 1500 rpm for 15 min at +4 °C. Serum was transferred into dry clean tubes and stored until analysis at −20 °C for no more than 5 days.

Thirty ml of blood was collected in Vacuette K3EDTA tubes (Greiner Bio-One) and processed on the same day in accordance with a recently published protocol [10 (link)]. In short, the blood was divided into two equal parts to enable comparison of single-step and two-step enrichment of CTCs. Using the single-step protocol (PX6.5), Parsortix® was the sole enrichment step employing the GEN3D6.5 microfluidic cassette with the critical gap size of 6.5 µm. With the two-step protocols, the blood samples were first enriched by DG centrifugation using 15 ml Percoll (GE Healthcare; d = 1.065 g/ml, 305 mOsm/kg), and then, the cell suspensions were further processed with GEN3D10 cassettes applying 23 mbar pressure (DG10), or GEN3D6.5 cassettes applying 99 mbar pressure (DG6.5). An overview on the respective protocols is given in Fig. 1. After the microfluidic separation, the captured cells were harvested and immediately lysed by adding 350 µl RLT lysis buffer (Qiagen).Fig. 1

Flow diagram of the protocols applied for the enrichment of circulating tumor cells (CTCs) and the detection of CTC-related gene transcripts. CTCs were enriched using the microfluidic Parsortix® enrichment alone and in combination with an upstream density gradient centrifugation. CTC-related gene transcripts were detected using Taqman® and Lightcycler technology (LC)

The study was performed according to good practices of animal handling, with the approval of the Institutional Animal Care and Use Committee (ORBEA 13/2018) and the procedures were performed by licensed users of Federation of Laboratory Animal Science Associations—FELASA, conformed to the guidelines from the Directive 2010/63/EU of the European Parliament for the Protection of Animals Used for Science Purpose. 12-week-old Wistar rats from our breeding colony (Faculty of Medicine, University of Coimbra) were fed a specific volume (4 mL) of each fruit juice sample or the same volume of matched sugary solution (Figure 1B,C). Glycaemia was determined using a glucometer and reactive test stripes (Contour Next, Bayer Portugal, Lisboa, Portugal) before and 15, 30, 60, 90 and 120 min after gavage (n = 12/condition). In a different set of animals, the same protocol was followed, and blood samples were collected from the tail vein to Vacuette K3EDTA tubes (Greiner Bio-one, Kremsmünster, Austria) for evaluation of total antioxidant capacity. Blood samples were immediately centrifuged (2200× g, 4 °C, 15′) and the cellular fraction was diluted in the same volume of ultrapure H₂O and submitted to repeated cycles of freeze/thawing. Supernatant was stored at −80 °C and later used for the Total Antioxidant Capacity Assay Kit (ab65329, Abcam) according to the manufacturer’s instructions.

Rat sample blood was taken in VACUETTE K3-EDTA tubes (Greiner Bio-One, Austria) and RBC mass was purified via centrifugation at 1500 rpm for 15 min at +4 °C. Then, 5 µL of the cell suspension was transferred at room temperature to tubes containing 2.5 mL each of graded concentrations of NaCl (0.25, 0.35, 0.45, 0.55, 0.65%). The tubes were gently mixed and after incubation for 10 min at room temperature, the non-lysed red cells were removed by centrifugation at 1500 rpm for 15 min at +4 °C.
The relative amount of hemoglobin released into the supernatant was determined spectrophotometrically (Beckmann Coulter DU 800, USA) at 541, 555, 577 nm, with a 0.85% NaCI sample serving as a blank and a 0.1% NaCl sample serving as the 100% lysis point [20 (link)].

To detect individuals with primary adult-type lactose malabsorption, LCT genotyping (C/T₋₁₃₉₁₀ polymorphism) was performed [12 (link)]. VACUETTE® K3EDTA tubes (2 mL) (Greiner Bio-One International GmbH, Kremsmünster, Austria) were drawn from patients for genomic DNA extraction on a MagNA Pure Compact Instrument (Roche Diagnostics, Rotkreuz, Switzerland). Real-time PCR with specific fluorescent labelled hybridization probes, followed by a melting curve analysis (LCT T-13910C ToolSet™ (Roche Diagnostics)), was established on a LightCycler® 2.0 Instrument (Roche Diagnostics).
A fructose breath test protocol was established to detect patients with fructose malabsorption. Gas chromatography was employed to measure the H₂ and CH₄ concentration using a QuinTron Model DP Plus MicroLyzer™ (QuinTron, Milwaukee, WI, USA). After determining baseline breath H₂/CH₄ concentrations, fructose was given in a dose of 25 g dissolved in 200 mL of water. The end-expiratory breath H₂/CH₄ concentrations were measured at 15, 30, 45, 60, 75, 90, and 120 minutes after fructose ingestion. Patients were classified with fructose malabsorption, if a H₂ and/or CH₄ increase >20 ppm above baseline concentrations was observed [11 (link), 12 (link), 20 (link)].

Body weight and caloric intake were monitored during the treatment. Fasting blood glucose (6 h) were determined in blood from the tail vein. The intraperitoneal insulin tolerance test (ipITT) was performed after a 6 h fast using insulin per body weight (250 mU/kg) and the evaluation of glycaemia was performed at 0, 15, 30 and 60 min using a glucose meter and test strips (Accu-Chek Aviva, Roche, Basel, Switzerland). The area under the curve (AUC) was calculated. After treatment, animals were anesthetised and serum and plasma were collected to Vacuette K3EDTA tubes and Vacuette Z Serum clot activator tubes (Greiner Bio-One, Kremsmünster, Austria) for the measurement of plasma insulin and non-esterified fatty acids using the Rat Insulin ELISA Kit (Mercodia, Uppsala, Sweden) and the Serum/Plasma Fatty Acid Kit (Zenbio, Durham, NC, USA). After blood sample collection, animals were sacrificed by cervical displacement and the following tissues were collected: aorta, epididymal adipose tissue (EAT), liver, heart and kidney. Aorta was excised and used for functional studies, and EAT and liver sections, used for colorimetric staining, were immersed in 10% formalin and included in paraffin. Aorta, kidney and liver sections for fluorescence studies were preserved in OCT cryo-embedding media (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C.