Heparin

An animal-free human whole blood model of endotoxemia was used to investigate the effect of LPS exposure on the PAF-induced response of neutrophils, which was described previously in detail (36 (link)). In brief, 0.5 IU/ml heparin (B. Braun Melsungen AG, Melsungen, Germany) and 100 ng/ml LPS (#L2630, Merck) or PBS (control) were added to 9 ml blood, which was transferred into a Cortiva BioActive Surface (Medtronic, Meerbusch, Germany) coated tubing system using a coated connector (Medtronic) to interlink both ends. Following 1 h of rotation (3 rpm) on a spinning wheel (Snijders Labs) at 37°C, the blood was transferred to citrate anti-coagulated monovettes (Sarstedt). Neutrophil activation markers were analyzed directly in whole blood as described previously (36 (link)) with or without exposure to PAF (1 μM, 15 min, 37°C). The remaining whole blood was processed to isolate and to analyze neutrophils as described above.

The protocols of this study were approved by the Institutional Animal Care and Use Committee of the National Health Research Institutes, Taiwan. C57BL/6 and BALB/c nude mice were purchased from the National Laboratory Animal Center, Taiwan. C57BL/6 mice were implanted with 250 μL Matrigel (Corning) containing 100 ng/mL VEGF (Sigma-Aldrich) and 20 U/mL heparin (B. Braun Melsungen AG, Melsungen, Germany) with or without 25 μg/mL cordycepin for seven days. The level of hemoglobin was measured using Drabkin’s reagent (Sigma-Aldrich). For in vivo tumor xenograft experiments, BALB/c nude mice were subcutaneously injected with 2 × 10⁶ Huh-7 cells. Micro-osmotic pumps (ALZA; Palo Alto, CA, USA) with in vivo continuous delivery [48 (link),49 (link)] containing DMSO or cordycepin (2.4 mg/kg/day) were implanted subcutaneously into nude mice on day 18. Tumor volume was determined by sequential caliper measurement of the length (L) and width (W), calculated as LW²/2.

Leukemia progression was evaluated by weekly quantification of peripheral blood circulating tumour cells. Blood (70–100 µL) was collected from facial vein and stored in tubes containing 10 µL of Heparin (500un/mL, B.Braun, Germany). In each blood sample, a defined amount of beads (Coulter CC Size Standard L10) was added for absolute cell quantification and Red Cells Lysis Buffer 1x(RBC Lysis Buffer 10x, Biolegend, USA) was added in multiple rounds until complete erythrocyte clearance. Cells were stained with LIVE/DEADTM dead cell staining kit (InvitrogenTM, NY, USA) for 15 min at 4 °C. Total GFP + Live/Dead– cells were quantified by Flow cytometry on an LSR FortessaII Cell Analyzer (BD Biosciences). Data was analyzed with FlowJo X 10.0.7 software (TreeStar, USA) and the results shown as the absolute numbers per mL of blood. Animals were assigned randomly to treatment groups upon reaching a minimum level of 100 GFP+ CD45+ viable cells/mL of blood (treatment threshold).

Mice were deeply anesthetized by intraperitoneal injection of pentobarbital (150 mg/kg body weight, Streuli Pharma AG). Animals were perfused with isotonic Ringer solution (containing 5 ml/l Heparin, B. Braun) followed by paraformaldehyde (PFA, 4%, in 0.2 M phosphate buffer, pH 7). For immunohistological analysis, brains were removed and 4 h post-fixed in 4% PFA, transferred to 30% sucrose for cryoprotection and stored at 4 °C. Coronal sections with a thickness of 40 µm were cut using a sliding microtome (Microm HM430, Leica). Sections were collected and stored as free-floating sections in cryoprotectant solution at −20 °C until further processing. For spectrophotometric analysis of CNS tissue was isolated and stored at −20 °C before further processing.

Female German Landrace pigs with 55,2 ± 1,9 kg body weight (BW, mean ± SEM) from a disease-free barrier breeding facility were housed in fully air-conditioned rooms (22 °C room temperature, 50% relative humidity) and allowed to acclimatize to their surroundings for a minimum of seven days and fasted for 12 h before surgery with free access to water. The animals were premedicated with 8 mg/kg BW azaperone (Stresnil, Janssen-Cilag GmbH, Neuss, Germany), 15 mg/kg BW ketamine (Ceva GmbH, Duesseldorf, Germany) and 10 mg atropine (1 ml/1% atropine sulfate, Dr. Franz Köhler Chemie GmbH, Bensheim, Germany) administrated intramuscularly. After cannulation of the femoral vein, 600 mL of venous blood for the ex vivo perfusion of the kidneys was withdrawn into sterile blood bags filled with 5,000 IU of heparin each (B. Braun Melsungen AG, Melsungen, Germany). The animals were thereafter euthanized by an IV administration of 1 mL/kg BW pentobarbital (Narcoren, Merial GmbH, Hallbergmoss, Germany). After cardiac arrest, a midline laparotomy was performed and both kidneys were explanted simultaneously to achieve an equal warm ischemic time. The duration from cardiac arrest until explantation was recorded and regarded as warm ischemic time. In compliance with the 3R principle, other organs of the animals were retrieved for different in-house research purposes.

Animals were deeply anesthetized by intraperitoneal injection of pentobarbital (150 mg/kg bodyweight) and subsequently transcardially perfused with isotonic Ringer solution (5 ml/l Heparin, B. Braun) and 4% Paraformaldehyde (PFA, in 0.2 M phosphate buffer, pH 7.4). Brains were removed from the skulls and post-fixed for 4 h at 4°C. For cryoprotection, brains were transferred to 30% sucrose and kept overnight at 4°C. Coronal sections with a thickness of 40 μm were cut using a sliding microtome (Microm HM430, Leica). Sections were collected and stored as free-floating sections in cryoprotectant solution at −20°C until further processing. For immunohistochemical staining brain sections were washed with 0.1 M phosphate buffer (PB) and then incubated with a blocking and permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum) for 30 min at RT shaking. Sections were incubated with primary antibody (rat-CD31, BD Biosciences, 1:100) overnight at 4°C. The next day sections were washed and incubated with corresponding secondary antibodies for 2 h at RT. All antibodies were diluted in blocking and permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum). Nuclei were counterstained with DAPI (1:2000 in 0.1 M PB). Sections were mounted in 0.1 M PB on Superfrost PlusTM microscope slides (Thermo Fisher) and coverslipped using Mowiol.

Animals were euthanized using pentobarbital (i.p, 150 mg/kg body weight, Streuli Pharma AG) and perfused transcardially with Ringer solution (containing 5 ml/l Heparin, B. Braun) followed by paraformaldehyde (PFA, 4%, in 0.2 M phosphate buffer, pH 7). Brain tissue was collected and post-fixed for 6 h in 4% PFA. For cryoprotection, tissue was transferred to 30% sucrose and stored at 4°C. Coronal sections were cut at a thickness of 40 µm using a sliding microtome (Microm HM430, Leica), collected, and stored as free-floating sections in cryoprotectant solution at −20°C.
For immunostaining, brain sections were blocked with 5% normal donkey serum for 1 h at room temperature and incubated with primary antibodies (rabbit anti-GFAP 1:200, Dako, #GA524; goat anti-Iba1, 1:500 Wako, #011-27991; NeuroTrace™ 1:200, Thermo Fischer; mouse anti-NeuN Antibody 1:500, Merck, #MAB377; rabbit anti-Neurofilament 200 antibody 1:200, Merck, #N4142; guinea pig anti-Neurofilament L, 1:200, Synaptic Systems, rat anti-CD31 antibody 1:50, BD Biosciences, #MEC13.3; goat anti-CD13, 1:200; R&D Systems, #AF2335) overnight at 4°C. The next day, sections were incubated with corresponding secondary antibodies (1:500, Thermo Fischer Scientific). Nuclei were counterstained with DAPI (1:2,000 in 0.1 M PB, Sigma). Mounting was performed using Mowiol.