Vacuette k3edta

Whole blood was collected from 15 individual healthy male and female blood donors (36 ± 10 years old) after written informed consent was given. HAS was prepared by centrifuging whole blood onto 9 mL silicon coated blood collection tubes (VACUETTE^® z serum clot activator, Greiner bio-one, Kremsmünster, Austria) at 1770 g for 10 min. The top layer containing the supernatant was removed and the resulting fibrin clot (middle layer) was separated from the tube by discarding the bottom part containing red blood cells. The fibrin clot was gently squeezed with a non-absorbable sterile material in a petri dish to extrude HAS. HAS was pooled from the individual 15 blood donors and stored at −80 °C. Leukocyte poor PRP (lpPRP) was prepared by transferring whole blood from the same donors onto 9 mL EDTA coated blood collection tubes (VACUETTE^® K3EDTA, Greiner bio-one, Kremsmünster, Austria) and centrifuged at 440 g for 10 min. The platelet enriched plasma (middle layer) along the poor platelet plasma (top layer) was further transferred to 15 mL falcon tubes leaving the leukocytes and RBC region and secondary centrifugation at 1770 g for 10 min was performed. The resulting lpPRP was pooled from individual donors and stored at −80 °C. Pooled lpPRP enclosed on average 1 × 10⁶ platelets/mL.

All animals were deeply anesthetized with urethane. Exsanguination (via transcardial puncture) and a bilateral pneumothorax were then performed as secondary confirmation for euthanasia. For the time-course rat study: blood was collected via cardiac puncture into EDTA tubes (Greiner Bio-One™ VACUETTE® K3EDTA, Kremsmünster, Austria) and immediately placed on ice. Tubes were centrifuged at 699 × g and 4 °C for 15 min. Plasma was aliquoted and stored at −80 °C. A piece of LV apex (unaffected by the cardiac puncture) was sectioned and submerged into RNAlater^TM solution (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) at 4 °C for ≥24 h for RNA stabilization prior to storage at −80 °C for gene expression analysis. To obtain a cross-sectional cut of the heart at mid-ventricular level for histology, a transcardial perfusion was performed with phosphate-buffered saline and then with 4% paraformaldehyde for fixation. The left femur was dissected for standardization purposes of cardiomyocyte dimensions. For the minocycline study: a transcardial perfusion (same as above) was performed for fixation and collection of the RVLM to assess sympatho-excitatory neurons.

For this experiment, blood samples collected for a previous study were utilized [18 (link)]. In short, 12 Holstein–Friesian calves (7 female, 5 male) were blood sampled weekly via jugular venipuncture for the first month of life (7 ± 0.8, 14 ± 1.0, 20 ± 0.8, and 29 ± 0.8 days of age). Blood was collected into plain vacuum tubes (BD Vacutainer; Becton, Dickinson and Company, Plymouth, UK) for serum collection. Blood was allowed to clot at room temperature, and sera were subsequently harvested after centrifugation at 2000× g for 20 min at 4 °C, aliquoted, and stored at −80 °C pending analysis within 2 months of collection. Blood from each calf was also collected in EDTA vacuumed tubes (Vacuette K3EDTA; Greiner Bio-One GmbH, Kremsmünster, Austria), immediately stored on crushed ice, and transported to the laboratory for isolation of peripheral blood mononuclear cells (PBMCs). The husbandry management of these calves is detailed in the study of the origin [18 (link)].

Venous blood samples were collected from the cephalic vein (or antecubital vein) of the healthy volunteers. Serum and plasma were collected in special pre-vacuumed containers (Vacuette® K3 EDTA and Vacuette® plus clot activator, Greiner Bio-One, Austria). Blood was stored at 4°C for 3 h. Samples were then centrifuged at 2000 g for 10 minutes at 4°C and then immediately aliquoted and stored at -80°C until further analysis.