S monovette k3 edta

Plasma samples were obtained from intensive care patients suffering from COVID-19 who required mechanical ventilation (Department of Internal Medicine, Hospital St. Vinzenz, Zams, Austria) between November 2020 and January 2021 (12 patients; time course; 134 samples obtained in total; the results of the main study regarding platelet-monocyte complexes in these samples has been published elsewhere^{46 (link)}). Sample collection was approved by the Ethics Committee of the Medical University of Innsbruck (Reference number 1144/2020, date of approval: May 20, 2020). The study was conducted in accordance with the declaration of Helsinki and guidelines of good clinical practice as well as local standard operating procedures. Freshly drawn whole blood (S-Monovette® K3 EDTA, Sarstedt, Nümbrecht, Germany) was centrifuged at 2000×g (15 min, 22 °C), and plasma was stored at – 80 °C until flow cytometric analysis of EVs (see below).

At 5.5 months after the heterologous primary vaccination, and 2 weeks (antibody titers peaked around 14–19 days post initial heterologous vaccination (25 (link))) after the BNT162b2 ‘booster’ (7 months post primary vaccination), blood was drawn into S-Monovette^® Serum Gel (Sarstedt) or S-Monovette^® K3 EDTA tubes. Serum gel collection tubes were centrifuged at 1,500 × g at 20°C for 15 min, aliquoted, and stored at -20°C until further use. PBMCs were obtained from EDTA tubes using density gradient centrifugation by Pancoll human (Pan Biotech, Germany), and erythrocytes were removed by ACK lysis buffer (Lonza, Walkersville, MD, USA). Mononuclear cells were counted for viability using a Countess II Automated Cell Counter (Thermo Fisher) with trypan blue stain and were cryopreserved in aliquots of up to 1 × 10⁷ cells in 10% DMSO in heat-inactivated FCS.

Venous blood for the analysis of nitrosative and carbonyl stress parameters was collected from COVID-19 patients, convalescents, and healthy controls into S-Monovette K3 EDTA and S-Monovette®® tubes (Sarstedt, Germany). Blood was collected in a fasting state, and the participants had not performed strenuous physical activity for 24 h before the test. The collected samples were immediately centrifuged at 4000×g for 10 min at a temperature of + 4 °C (MPW 351, MPW Med. Instruments, Warsaw, Poland). The plasma and the serum were separated from morphotic elements and protected against oxidation (10 µL of 0.5 M BHT/1 mL of serum/plasma). The separated blood components were stored at a temperature of − 80 °C until analysis, but not longer than for six months.

At the times of collection (0 h and after 1/3 days, 1 days, 2 days, 7 days, 14 days, 28 days), the fishes were anesthetized with etomidate solution and around 2 mL of whole blood was taken from the caudal vein using S-Monovette K3 EDTA (Sarstedt). Then, the fish were killed and the brain was dissected out and preserved in RNAlater (Sigma-Aldrich). After collection, the blood was mixed by gently inverting the tube several times and immediately centrifuged at 4000×g at room temperature for 5 min. The plasma layer (approximately 400 μL) from the top of the tube was transferred into a fresh tube. Plasma was stored at − 20 °C during sample collection, shipped on dry ice, and stored at − 80 °C in the laboratory until next procedures.