Whole blood samples were taken into vacutainer tubes containing 3.2% trisodium citrate (Greiner Bio-One, Stonehouse, UK) and ethylenediaminetetraacetic acid (BD, Oxford, UK). Samples were taken before heparin administration and 30 min after reversal of heparin by protamine sulfate, PPP was prepared by centrifuging samples twice at 1650g before freezing in aliquots at −80°C for testing later. Full blood cell counts were performed on an ABX Pentra DX 120 automated analyser (Horiba Medical, Northampton, UK). The PT, APTT, Clauss fibrinogen and factors II, V, VII, VIII, IX, X, XI, antithrombin, protein C, free protein S and postoperative anti-Xa activity were measured on an ACL 500 Top (Instrumentation Laboratory, Cheshire, UK) automated coagulometer using standard manufacturer protocols and reagents. Factor XIII activity was measured in a flat-bottomed Immulon 2hb 96-well plate (Diagnostica-Stago, Asnières sur Seine, France) using a chromogenic assay kit from Technoclone (Vienna, Austria) and light absorbance was measured using a plate reader (BioTek, Winoosi, Vermont, USA).
Trisodium citrate
Manufactured by Greiner
Sourced in Austria, United Kingdom
Trisodium citrate is a powdery, water-soluble salt that is commonly used as a buffering agent in laboratory settings. It helps maintain a specific pH level in solutions.
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Lab products found in correlation
10 protocols using trisodium citrate
1
Coagulation Markers in Heparin Reversal
2
Virally Suppressed PLHIV Plasma Preparation
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We previously described the population investigated in this (17 (link)). The study was approved by the local ethics committee (CMO Arnhem-Nijmegen, The Netherlands; NL425561.091.12, 2012/550). Virally suppressed PLHIV and uninfected controls were enrolled in the study after obtaining written informed consent. Participants were excluded if they had either an active hepatitis B or C infection, if they had signs of other acute infections or if they had received coumarin derivates or direct oral anticoagulants. Blood was collected into vacuum tubes (1 volume 0.109 mol/L trisodium citrate to 9 volumes blood; Greiner Bio-One). Platelet poor plasma (PPP) was prepared by centrifugation at 2840 g for 10 minutes and stored at -80°C.
3
Platelet-poor Plasma Extraction Protocol
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Whole blood was obtained on admission immediately after vascular access was available and again 12 h later and stored in blood collection tubes containing 3.8% trisodium citrate (Greiner BioOne, Kremsmünster, Austria). Immediately thereafter, samples were centrifuged for 10 min at 3000 g and platelet poor plasma was stored at −80 °C until final analysis.
4
Isolation of Platelet-Rich and Platelet-Free Plasma
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Blood from fasted volunteers was obtained by venepuncture from the median cubital vein using a 19G butterfly needle. The first 5 ml of collected blood was discarded. The blood to be used in this study was drawn into a vacutainer containing tri-sodium citrate (3.2%; Greiner Bio-One, UK) and processed within 30 min. To obtain platelet free plasma (PFP), blood was centrifuged at 1500×g for 15 min and the upper two thirds were collected and centrifuged again at 13,000×g for 2 min. The upper three quarters from each tube were collected and identified as PFP.
Blood was centrifuged at 175×g for 15 min to obtain platelet-rich plasma (PRP). Platelets were then isolated by further centrifugation (1000×g, 10 min) in the presence of prostacyclin (PGI2, 1 µg/ml; Sigma, UK). The resulting pellet was washed in modified Tyrode’s (MTH) buffer (containing 134 mM NaCl, 2.9 mM KCl, 0.34 mM Na2HPO4, 12 mM NaHCO3 and 1 mM MgCl2; pH 7.4) containing HEPES (20 mM; Sigma, UK) and re-suspended in an equal volume of MTH. Platelets were counted and adjusted to a final working concentration of 3 × 108 platelets/ml.
Blood was centrifuged at 175×g for 15 min to obtain platelet-rich plasma (PRP). Platelets were then isolated by further centrifugation (1000×g, 10 min) in the presence of prostacyclin (PGI2, 1 µg/ml; Sigma, UK). The resulting pellet was washed in modified Tyrode’s (MTH) buffer (containing 134 mM NaCl, 2.9 mM KCl, 0.34 mM Na2HPO4, 12 mM NaHCO3 and 1 mM MgCl2; pH 7.4) containing HEPES (20 mM; Sigma, UK) and re-suspended in an equal volume of MTH. Platelets were counted and adjusted to a final working concentration of 3 × 108 platelets/ml.
5
Healthy Volunteer Blood Donation
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Blood was donated by healthy volunteers that were free from anticoagulant or antiplatelet medication for a minimum period of 4 weeks. Studies were approved by the local Medical Ethics Committees (Maastricht University Medical Centre). All donors provided full informed consent in accordance with the Declaration of Helsinki, and procedures were in accordance with the local regulations and guidelines. Blood drawing was by venipuncture using a vacuum container. The blood was collected into a 9 mL tubes with 3.2% trisodium citrate (Greiner, Alphen a/d Rijn, The Netherlands). For all the microfluidics studies, collected blood was stored at room temperature and used within 4 h.
6
Blood Collection and Plasma Isolation
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Human blood was collected by a qualified phlebotomist and handled using appropriate safety procedures, and the Perron Institute approved the procedure. Blood was obtained from one of the authors (BM) via venepuncture into trisodium citrate (3.2% w/v final) vacuum collection tubes (Greiner Bio One, Austria). Plasma was obtained from whole blood in collection tubes after centrifugation for 5 min at 3500 rpm. Whole blood and plasma were stored at 4 °C before use and used on the day of collection or 1 to 2 days after collection for thrombolysis studies.
7
Plasma EV Isolation and Characterization
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This study was conducted in accordance with the Declaration of Helsinki and approved by the local ethics committees. Informed consent was obtained from all volunteers. Blood samples were collected from healthy human volunteers. Blood was drawn from the antecubital vein by a qualified medical technologist using a butterfly catheter with a 21-gauge needle. After discarding the first 3 ml, blood was collected in trisodium citrate (3.2%) (Greiner Bio-One, Frickenhausen, Germany). Immediately after collection samples were centrifuged twice at 2,500×g for 15 min at 20°C, in order to prepare platelet depleted plasma. The supernatant (plasma) was collected, with approximately 10 mm and 4 mm distance to the pellet, following the first and second centrifugation steps, respectively. Samples were frozen and stored at −80°C.
Plasma aliquots (1.1 ml) were shipped as frozen vials to the 6 trial participants. Vials were stored at −80°C and measured within 24 h after thawing. Before TRPS measurement EV samples were centrifuged at 2,000×g for 10 min (4°C if possible) to remove any aggregates formed during the freeze–thaw process. After centrifugation, 500 µl of the resulting supernatant was loaded on a qEV column (see below) for further EV purification.
Plasma aliquots (1.1 ml) were shipped as frozen vials to the 6 trial participants. Vials were stored at −80°C and measured within 24 h after thawing. Before TRPS measurement EV samples were centrifuged at 2,000×g for 10 min (4°C if possible) to remove any aggregates formed during the freeze–thaw process. After centrifugation, 500 µl of the resulting supernatant was loaded on a qEV column (see below) for further EV purification.
8
Analyzing SLE-Induced Fibrin Property Alterations
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The data for set 4 came from a study on the effect of systemic lupus erythematosus (SLE) on fibrin properties [24 (link)]. Twenty-eight patients, with 82/18% female/male ratio at a mean age of 37 ± 13 years, were included in the original study, based on the revised criteria of the American College of Rheumatology for SLE. Informed consent was obtained from the SLE patients and healthy donors under the approval of the Ethical Committee of the Kazan State Medical University (Kazan, Russia; protocol #2 as of 21 February 2017). A subset of all available SEM images from this study was included in the current study. Blood was drawn under aseptic conditions without venous stasis using vacutainers containing 3.8% trisodium citrate (Greiner Bio-One, Kremsmünster, Austria) at 9:1 v/v. For investigations of plasma clots, the blood was centrifuged at room temperature for 15 min at 1500× g to obtain platelet-poor plasma, then 5 min at 10,000× g to obtain platelet-free plasma, which was then aliquoted and stored at −80 °C until further analysis. For control experiments, platelet-free plasma from at least 10 healthy donors was pooled, aliquoted, and stored at −80 °C until use. The frozen samples were thawed at 37 °C for 60 min and used within 2 h.
9
Blood Plasma Preparation Protocol
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Blood was drawn under aseptic conditions without venous stasis using vacutainers containing 3.8% trisodium citrate (Greiner Bio-One, Austria) at 9:1 v/v. For investigations of plasma clots, the blood was centrifuged at room temperature for 15 min at 1,500 g to obtain platelet-poor plasma, then 5 min at 10,000 g to obtain platelet-free plasma, which was then aliquoted and stored at −80°C until use. For control experiments, platelet-free plasma from at least 10 healthy donors was pooled, aliquoted and stored at −80°C until use. The frozen samples were thawed at 37°C for 60 min and used within 2 h. A part of each individual blood sample was used for standard immunological, hematological, and biochemical tests.
10
Platelet Isolation and Plasma Preparation
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For platelet isolation, blood was drawn from an antecubital vein into 3.5 mL vacuum tubes containing 0.129 mM trisodium citrate as anticoagulant (Greiner Bio-One, Kremsmünster, Austria). To obtain platelet rich plasma (PRP), two 1 mL aliquots of citrated blood in 1.5 mL tubes were centrifuged [8 min, 67 g, room temperature (RT)] and the supernatant PRP was pooled into a fresh 1.5 mL tube. Platelets were pelleted (2 min, 2,000 g, RT) in the presence of 0.8 μM PGI2 (Sigma-Aldrich, St. Louis, MO, USA) and washed once in phosphate-buffered saline (w/o: Ca2+ and Mg2+) containing PGI2 (0.8 μM). The supernatant was carefully discarded and the platelet pellet was frozen at −80°C until further processing.
For plasma preparation citrated blood was centrifuged (10 min, 1,000 g, 4°C) to separate the cellular fraction and the plasma supernatant, which was subsequently cleared of debris by a second centrifugation step (10 min, 10,000 g) and stored at −80°C.
For plasma preparation citrated blood was centrifuged (10 min, 1,000 g, 4°C) to separate the cellular fraction and the plasma supernatant, which was subsequently cleared of debris by a second centrifugation step (10 min, 10,000 g) and stored at −80°C.
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