S monovette serum gel

Venepuncture was carried out among all subjects in a fasting state (min. 10 h) in the morning of various days. After a 30-min storage period, the filled blood tubes (S-Monovette® serum gel by Sarstedt®) were centrifuged for 10 min at 2000 x g in a Sarsted® LC-6 centrifuge and immediately stored at − 18° Celsius in a lightproof storage unit.
After completion of the voyage and in compliance with the cold chain, the following blood parameters were determined in a German laboratory (Lademannbogen GmbH, Hamburg, Germany): cholesterol, HDL, non-HDL-cholesterol, LDL, LDL/HDL ratio, triglyceride, alkaline phosphatase, uric acid and fasting glucose. The analysis of all results referred to reference values from German population studies as no reference values were available for Kiribati in the literature.

Bilateral ovariectomy was performed under general anesthesia using a ventral
midline approach and approved standard methods. The success of the
ovariectomy was verified by measurement of serum 17 $\mathit{\beta }$ -estradiol
concentrations. All animals had 17 $\mathit{\beta }$ -estradiol concentrations
$<\mathrm{20.0}$   $\mathrm{pg}{\mathrm{mL}}^{-\mathrm{1}}$ after ovariectomy (data not shown). Body mass was
determined with a bench scale (FCB, Kern, Balingen–Frommern, Germany) and
blood samples were collected after the morning feed between 09:00 CET and
noon on the day before the quantitative computerized tomography measurement.
Blood samples were collected from the femoral vein via a syringe,
immediately transferred into a tube (S-Monovette^® Serum-Gel,
Sarstedt, Nümbrecht, Germany), centrifuged, and the serum was stored
frozen until analysis. The 17 $\mathit{\beta }$ -estradiol concentration was measured by
radioimmunoassay (RIA; DSL-43100; Diagnostic Systems Laboratories Inc., Webster, TX, USA).

The sampling of the peripheral blood was performed under the standard procedure from the cubital vein, using 7.5 mL tubes of S-Monovette^® Serum Gel (Sarstedt) preoperatively, once or twice during the follow-up period and always when recurrence was suspected. The samples were transported to the Department of Clinical Biochemistry UH Brno, where the serum was separated by centrifugation, and the samples were analyzed either immediately (CA125, HE4) or stored frozen at −80 °C until analysis (DJ1 and L1CAM).
The quantitative assessments of L1CAM and DJ1 levels were performed by enzyme-linked immunosorbent assay (ELISA) using ELISA reader iMARK (Bio-Rad). For L1CAM, kit CN MBS 2023094 (MYBioSource, USA) was used. DJ1 serum levels were measured using kit CN CY-9050V2 (CircuLex MBL, UK). The serum concentrations of HE4 and CA125 were determined using quantitative, chemiluminescent microparticle immunoassay (CMIA) on the analyzer Architect i2000 (Abbott, Abbott Laboratories, USA). For CA125 measurement, the diagnostic set ARCHITECT Ca125 II (CN 2K45-24, Abbott) was used. HE4 serum level assessments were performed using the diagnostic set ARCHITECT HE4 (CN 2P51-25, Abbott).

Blood samples were collected in S-Monovette serum-gel vacutainers treated with clotting activator (Sarstedt) for serum preparation and in vacutainer cell preparation tubes (CPT) with sodium-citrate (BD Biosciences) for isolation of PBMCs. Serum was prepared from clotted blood by centrifugation for 10 min at 2500 × g and stored at −80 °C. For PBMC isolation, whole blood was centrifuged for 30 min at 1700 × g, and the PBMC layer was then transferred and washed twice with PBS. The pellet was re-suspended in 0.5 ml freezing medium (either 50% FBS and 50% RPMI 1640 or 12.5% HSA and 87.5% RPMI 1640) and the cell number was determined by trypan blue staining. Following addition of 0.5 ml DMSO medium (either 50% FBS, 30% RPMI 1640, and 20% DMSO or 12.5% HSA, 67.5% RPMI 1640, and 20% DMSO) dropwise, the cells were frozen immediately in a freezing container at −80 °C. After 24 h, PBMCs were transferred into liquid nitrogen.

Whole blood samples were taken on the first, fourth, and seventh days of antibiotic treatment. The first 24 samples were extracted in duplicate for screening of the selected miRNAs (see below). Concomitantly, the blood samples for NGAL determination were collected into 2.6 mL neutral tubes (S-Monovette® K3 EDTA, 2.6 mL, red, Sarstedt AG & Co. KG, Germany; or Vacuette® K3 EDTA 2 mL, violet, Greiner Bio-One GmbH, Germany) and centrifuged. After centrifugation, the plasma was aspirated, collected into cryotubes, and frozen at − 70 °C until use.
The blood samples for IL-6, PCT, and S_crea concomitantly with other standard care biochemical parameters also were collected (S-Monovette® serum-gel, 4.9 mL, brown, Sarstedt AG & Co. KG, Germany in University Hospital Ostrava; or Vacuette® serum-gel, 5.0 mL, red, Greiner Bio-One GmbH, Germany in University Hospital Olomouc) and analyzed immediately after centrifugation in both hospitals.

Pre-treatment peripheral blood samples from young patients were collected using S-Monovette^® Serum-Gel (SARSTEDT) tubes and processed according to the standard NCT biobank protocols. The blood samples were centrifuged at 2,500 × g for 10 min for serum separation, divided into 200–300 uL aliquots, and stored at −80°C until further analysis.