Hirudin blood tube

In vitro aggregation tests were conducted using freshly collected whole blood with Multiplate platelet function analyzer (Roche Diagnostics Polska Sp. z o.o., Warsaw, Poland), the five-channel aggregometer based on measurements of electric impedance. The Multiplate analyzer allows the duplicate measurement with dual electrode probes. Blood was drawn from carotid of rats with hirudin blood tube (Roche Diagnostic). 300 μL of hirudin anticoagulated blood was mixed with 300 μL pre-warmed isotonic saline solution containing studied compound in DMSO or DMSO (0.1% final) and pre-incubated for 3 min at 37 °C with continuous stirring. The agonists (ADPtest, COLtest, Roche Diagnostic) were diluted using isotonic sterile NaCl solution. Aggregation was induced by adding collagen (final concentration 1.6 µg/mL), or adrenaline and subthreshod concentration of ADP (final concentration 50 µM + 1.6 µM). Activated platelet function was recorded for 6 min. The Multiplate software analyzed the area under the curve of the clotting process of each measurement and calculated the mean values.
Data were presented as Mean ± SEM. Statistical comparisons were made by the analysis of variance (ANOVA) and significance of the differences between control group and treated groups was determined by Dunnet post hoc test. p < .05 was considered significant.

Three ml of venous blood was drawn from an antecubital vein using a vacutainer system before and at the fifth day of Omega-3 treatment period. The volunteers rested 30 min before sampling and had had no stress or physical exertion during the morning, which can increase platelet aggregation [23 (link)]. Blood was collected in a 3.0 ml Hirudin blood tube (Roche Diagnostics GMbH, Mannheim; Germany). The hirudin sprayed within the blood tube exerts its inhibitory effect on thrombin, without interfering with physiological calcium levels [24 ]. The blood samples were stabilized at room temperature for 30 min, followed by MEA analysis within 3 h as recommended by the manufacturer [24 ] and Würtz et al. [25 (link)] to reduce test variability.

GFP-E1162 and GFP-E1162ΔtirE from a blood agar plate were diluted in BHI to OD₆₀₀ 0.1 and grown at 37°C to OD₆₀₀ 0.4, washed with PBS, and resuspended in RPMI-HSA. Blood was obtained from a healthy volunteer in a hirudin blood tube (Roche Diagnostics, Switzerland) and diluted in RPMI-HSA. For all whole-blood experiments, blood was used at 80% if not otherwise indicated.
Phagocytosis was performed in round-bottom polystyrene tubes under rigorous shaking at 37°C for 20 min. Erythrocytes were lysed by adding FACS lysing solution (BD) for 20 min at 25°C. The remaining immune cells were washed with RPMI-HSA and fixed in 150 µl 1% paraformaldehyde in RPMI-HSA. Fluorescence was measured through flow cytometry (FACSCalibur; Becton Dickinson, USA).
For blood survival assays, E1162 and E1162ΔtirE were grown in BHI to OD₆₀₀ 0.4, washed with PBS, and incubated with 80% fresh hirudin blood in siliconized tubes (Sigma-Aldrich, USA) on a turning wheel at 37°C for 3 h, if not otherwise indicated. Blood cells were lysed in 0.3% saponin, and CFUs were counted on blood agar plates.