Blood samples from healthy volunteers or CD-1 mice were collected in tubes containing hirudin (Roche, Switzerland) as the anticoagulant to preserve complement activity. One hundred sixty microliters of freshly drawn blood was mixed with 20 μl volumes containing S. aureus (∼1 × 107 CFU/ml), S. haemolyticus (∼1 × 106 CFU/ml), or E. coli (∼1 × 107 CFU/ml) or 40 μl of L. lactis (∼1 × 108 CFU/ml) in RPMI 1640–HSA. When applicable, 20 μl of RPMI 1640–HSA or recombinant full-length SdrD (rSdrD) (4 (link)) in RPMI 1640–HSA was added to the samples in siliconized tubes (Sigma-Aldrich, Germany) at a final concentration of 12 μg/ml. After incubation for 3 h at 37°C on a horizontal rotator, the blood cells were lysed by addition of 1 ml ice-cold H2O supplemented with 0.3% saponin (Sigma-Aldrich, Germany). Bacterial survival was evaluated by serial dilution on blood or Todd-Hewitt broth (THB) agar plates. Percent survival was determined by comparing the number of surviving bacteria to the number of bacteria in the inoculum.
Hirudin
Manufactured by Roche
Sourced in Switzerland, Germany, Japan
Hirudin is a lab equipment product manufactured by Roche. It is a natural anticoagulant protein derived from the saliva of the medicinal leech Hirudo medicinalis. Hirudin functions as a direct thrombin inhibitor, binding to and inhibiting the enzymatic activity of thrombin, a key component in the blood coagulation process.
Automatically generated - may contain errors
Lab products found in correlation
Siliconized tubes, by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
Hirudin blood collection tube, by Roche (1 mentions)
Bsk h media, by Merck Group (1 mentions)
Histopaque 1077 and 1119, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by BD (1 mentions)
Sodium citrate, by Terumo (1 mentions)
9 protocols using hirudin
1
Bacterial Survival in Human Blood
2
Measuring Platelet Thrombus Formation
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Example 4
Blood collected in a vacuum blood collection tube containing low-molecular-weight heparin (final concentration, 2 IU/mL) and CTI (final concentration, 50 μg/mL), in a hirudin blood collection tube (manufactured by Roche Diagnostic; hirudin final concentration, 50 μg/mL), or in a BAPA blood collection tube (BAPA final concentration, 100 μM) was used to measure platelet thrombus formation using the device shown in
In every case, a vacuum blood collection tube having a butyl rubber stopper was used.
As a result, as shown in
3
Culturing and Preparing Borrelia hermsii
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Borrelia hermsii strain HS1 was a kind gift from prof. Bergström, University of Umeå, Sweden. Bacteria were cultured in BSK-H media (Sigma-Aldrich, Darmstadt, Germany) at +33° C in 5% CO2 and 100% humidity and number was determined by calculation under dark-field microscopy using 40x magnification. Prior to usage bacteria were pelleted (8,000 g 15 min at RT) and washed 3 times with PBS (phosphate-buffered saline; 120 mM NaCl, 30 mM phosphate, pH 7.4). Blood was drawn into hirudin (Roche Diagnostics, Mannheim, Germany) tubes from healthy human volunteers after informed written and signed consent (Ethical Committee decision HUS/135/2020, Hospital district of Helsinki and Uusimaa). The plasma was isolated by centrifugation. To obtain serum the blood was drawn into serum tubes, blood was allowed to clot, after which it was centrifugated and serum collected. Serum was kept at -80° C and heat-inactivated (+56° C, 30 min) to remove complement activity.
4
Isolation of Human Neutrophils
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Neutrophils were isolated as previously described [30 (link)] with minor modifications, as follows. Blood was drawn to tubes containing hirudin (Roche Diagnostics, Mannheim, Germany) from healthy human volunteers after informed written and signed consent (Ethical Committee decision 406/13/03/00/2015, Hospital district of Helsinki and Uusimaa). The blood samples were diluted 1:1 with PBS and centrifuged through a gradient (Histopaque® 1.119 and 1.077; Sigma-Aldrich, Steinheim, Germany) at 320 x g for 20 min at 22°C. The neutrophil layer was collected, washed once with RPMI 1640 (Gibco®) containing 0.05% HSA (RPMI-HSA), and the remaining red blood cells were lysed with ice-cold water. The isotonic conditions were reestablished with PBS and the cells were washed before diluted with RPMI-HSA.
5
Assessing Platelet Transfusion Efficacy
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This single-center observational study was approved by the Ethics Committee of Kagoshima University Hospital (approval no. 170178–2). The study was conducted in compliance with the Declaration of Helsinki, and written informed consent to participate was obtained from patients or their close relatives. We included 10 adult patients admitted to the ICU of Kagoshima University Hospital between November 2017 and October 2019 who required a platelet transfusion. We excluded patients administered drugs that affect the hemostatic system, such as antiplatelet agents or anticoagulants. The use of anticoagulants for the maintenance of arterial lines or extracorporeal devices was permitted. The need for platelet transfusion was determined in accordance with our standard clinical practice, independent of the present study.
Blood was drawn from the radial arterial line before and after platelet transfusion. The samples were anticoagulated with EDTA (Becton Dickinson Co., Fukushima, Japan), 3.2% sodium citrate (Terumo, Tokyo, Japan), or hirudin (Roche Diagnostics GmbH, Mannheim, Germany) and were used for blood cell counts, thromboelastometry, T-TAS, and multiplate aggregometry. After platelet transfusion, platelet concentrates remaining in the transfusion tube were collected and analyzed for their thrombogenic potential.
Blood was drawn from the radial arterial line before and after platelet transfusion. The samples were anticoagulated with EDTA (Becton Dickinson Co., Fukushima, Japan), 3.2% sodium citrate (Terumo, Tokyo, Japan), or hirudin (Roche Diagnostics GmbH, Mannheim, Germany) and were used for blood cell counts, thromboelastometry, T-TAS, and multiplate aggregometry. After platelet transfusion, platelet concentrates remaining in the transfusion tube were collected and analyzed for their thrombogenic potential.
6
Platelet Aggregation Analysis by Multiplate
7
Venous Blood Collection for Multimodal Analysis
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Venous blood was collected from ante-cubital vein aseptically via a butterfly 20-gauge needle into vacutainers, depending on the assay; EDTA; Na-citrate; Serum SST™ (all Becton Dickinson) and Hirudin (Roche Diagnostics). Serum SST™, Na-citrate and EDTA were utilised for haematological analysis. Na-citrate was also used for EV quantification and soluble P-selectin (CD62P) analysis. One EDTA was used for flow cytometric analysis. Hirudin was used for platelet aggregation analysis. The first 5 mL of blood before sampling was discarded to avoid excessive platelet activation.
8
Comprehensive Blood Collection and Analyses
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Blood was collected from the forearm using a 21-G needle and vacuum blood collection tubes after fasting for at least 6 h. Blood samples were anticoagulated with hirudin (Roche Diagnostics GmbH, Mannheim, Germany), ethylenediaminetetraacetic acid, or sodium citrate, which were used for T-TAS assays, complete blood cell counts, or coagulation assays, respectively. Serum samples were obtained from blood without anticoagulation, and were used for measuring levels of bilirubin, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, γ-glutamyl transpeptidase, albumin, globulin, creatine phosphokinase, amylase, glucose, low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglycerides, urea nitrogen, creatinine, uric acid, sodium, potassium, and chloride.
9
Precise Blood Collection for Analysis
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Before collection of each blood sample, a small volume of blood (~0.5 ml) was drawn from the sampling port and discarded to ensure the line was free of stagnant blood. Immediately afterwards, 8 ml of blood was collected into a sterile 10 ml syringe with 2 ml transferred into a collecting tube containing ethylenediaminetetraacetic acid (BD Vacutainer; ethylenediaminetetraacetic acid (EDTA); 1.8 mg/ml), 2 ml transferred into a collecting tube containing hirudin (Roche Diagnostics GmbH, Mannheim, Germany; 15 μg/ml), and 4 ml transferred into a collecting tube containing citrate (BD Vacutainer; 109 mol/m 3 ). These aliquots were then used for the different blood analyses described below. Following withdrawal of blood from the BCL that caused a volume loss, the resistances across the reservoirs were adjusted to maintain pressure and flow at baseline values.
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