Blood samples were obtained by venipuncture from healthy volunteers with no history of thrombosis or bleeding and who had not taken anti-inflammatory drugs for at least 10 days. In particular, samples dedicated to ECFC isolation were collected into heparin vacutainer tubes (BD Biosciences, San Jose, CA, USA), whereas samples perfused in thrombogenesis assay and used for platelet-rich plasma (PRP) preparation were collected in vacutainer tubes containing 0.109 M trisodium citrate as anti-coagulant (BD Biosciences, San Jose, CA, USA). All samples were processed and analyzed within 3 h after collection.
Trisodium citrate
Manufactured by BD
Sourced in United States, France
Trisodium citrate is a chemical compound used as a salt of citric acid. It is a white, crystalline powder that is soluble in water. Trisodium citrate is used as a buffering agent, preservative, and anticoagulant in various laboratory applications.
Automatically generated - may contain errors
15 protocols using trisodium citrate
1
Healthy Volunteer Blood Collection
2
Platelet-leukocyte interactions analysis
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Midstream flow urine was collected and stored for subsequent eicosanomic analysis.16 (link) Blood was collected by venepuncture into trisodium citrate (BD Diagnostics, UK). Platelet-rich plasma (PRP) was obtained by centrifugation at 175xg for 15 min. Platelet-poor plasma (PPP) was obtained by centrifugation of PRP at 12,000xg for 2 min. COX-1 protein presence was determined by western blotting in platelets and confocal microscopy in platelets and leukocytes. In addition, the number of platelet-monocyte and platelet-neutrophil aggregates were quantified to assess whether this variant modulates interactions of platelets with other blood cells using an ImageStream®X imaging flow cytometer (Merck Millipore, UK).
3
Platelet Collection and Preparation
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Peripheral blood samples were collected using a 21G needle into 0.109 M trisodium citrate (9:1 v/v, BD) from healthy volunteers with no exposure to drugs known to affect platelet function for at least 14 days prior to the study, as per standard practise. Platelet rich plasma (PRP) and platelet poor (PPP) was generated, with PRP used within 4 h of collection and PPP frozen at −80°C prior to use in assays.
4
Human Blood Collection and Consent
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Human blood was drawn from healthy donors of the Établissement Français du Sang Grand Est (EFS Grand Est, Nancy, France) who gave their informed written consent in accordance with the guidelines of the EFS Grand Est. Blood samples were collected using Vacutainer tubes containing 129 mmol/L trisodium citrate (BD Biosciences) purchased from Ozyme (Saint-Quentin-en-Yvelines, France). The study was conducted in accordance with the Declaration of Helsinki.
5
Healthy Adult Blood Sampling Protocol
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The study was given ethical permission from the National Medical Ethics Committee (number 82/07/14) and it conformed to the ethical principles given in the Declaration of Helsinki. Blood was obtained from 10 healthy adults (3 females, average age 32 ± 4 years and 7 males, average age 30 ± 5 years) with their written consent. Sampling was performed after at least 12 h of fasting. Each subject donated blood by venipuncture into two 2.7 mL vacutubes containing 270 μL 0.109 mol L−1 trisodium citrate (Becton Dickinson, Franklin Lakes, NJ, USA) and was gently mixed by turning the tube upside down multiple times. Prior to sampling, tubes were prewarmed to 37 °C and samples were kept in thermoblocks to minimize the activation of platelets caused by thermal stress [27 ,28 (link)]. Centrifugation of the samples started within 15 min after the acquisition of the first sample.
6
Platelet Function Assessment via VerifyNow and Aggregometry
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Blood samples were collected into Vacutainer tubes containing 0.109 mol/L trisodium citrate (Becton–Dickinson, Franklin Lakes, NJ) after overnight fasting. Anticoagulated blood was directly used for the VerifyNow® (VN) Aspirin assay (Accumetrics, San Diego, CA). Platelet rich plasma (PRP) separated by centrifugation (120g, 37 °C, 15 min) was used for AA induced platelet aggregation tests. Platelet depleted plasma (PDP), was obtained by two consecutive centrifugations (1500g, 25 °C, 20 min).
7
Rat Tissue Harvesting and Plasma Isolation
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On the day of sacrifice, rats were anesthesized with sodium pentobarbital (45 mg/Kg body weight, ip), blood was withdrawn from the abdominal aorta and collected in a vacutainer containing 9:1, v/v trisodium citrate (Becton Dickinson Co, Rutherford, NJ, USA), and plasma was immediately separated by a 10-min centrifugation (500× g at 4 °C). The liver, heart, kidneys, spleen, brain, ovaries or testis were harvested, washed in 0.9% saline solution, weighed and stored at −80 °C until analysis.
8
Platelet-Depleted Plasma Preparation
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Venous blood (blood/citrate ratio 9:1) was collected from 20 healthy individuals (10 males) in 0.109 mol/L trisodium-citrate (Becton Dickinson, San Jose, CA) with informed consent and as approved by the medical ethical committee of the Amsterdam UMC (W18-289-18335). Platelet-depleted plasma was prepared by two consecutive centrifugation steps for 20 min at 1560g at room temperature, first the blood and then the plasma layer, to prepare platelet-depleted plasma. The plasma was aliquoted, snap-frozen with liquid nitrogen for 15 min and stored at −80°C until use. Our procedure to prepare platelet-depleted plasma differs slightly from the protocol of Lacroix et al. [5 ], which will be discussed in the Discussion section.
9
Preparation of Platelet-Free Plasma
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Blood samples were obtained by traumatic puncture of the antecubital vein, using a 20-gauge needle, and placed into siliconized vacutainer tubes containing 0.129 mol/L trisodium citrate (from Becton and Dickinson France) as anticoagulant, in a ratio of nine parts of blood to one part of citrate. Platelet poor plasma (PPP) was obtained after double centrifugation of citrated whole blood for 20 minutes at 2000 g. Platelet-free plasma was prepared immediately after blood sampling using a 2-step centrifugation procedure: initially at 1500 g for 15 minutes at 20°C to prepare platelet rich plasma and then at 13000 g for 2 minutes at 20°C to prepare PFP. Samples were aliquoted and frozen at −80°C until assayed. All measurements were done in thawed plasma samples. All PPP samples were from vein punctures performed for routine evaluation of blood coagulation tests. Blood anticoagulated with EDTA was used for the determination of complete blood count. This study was approved by the ethics committee of Tenon University Hospital and was performed in accordance with the principles embodied in the Declaration of Helsinki.
10
Anticoagulant Plasma Levels in DOAC Patients
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Altogether ninety-six patients were recruited from three coagulation centres in the region of Stockholm (Danderyd Hospital, Karolinska University Hospital, and Stockholm Heart Centre) between 2013 and 2014. All patients received anticoagulant treatment according to clinical routine care. DOAC treatment included dabigatran (n = 23), rivaroxaban (n = 26), and apixaban (n = 20). The doses for each drug were chosen in line with the summary product characteristics (SPC) for DOACs, and all study patients reported full compliance during the last three days before trough plasma samples were collected. Patients on PT-INR guided warfarin therapy (n = 27) were included for comparison (VTE patients were included at least one month after any thrombotic event).
Blood samples were drawn by direct venipuncture from an antecubital vein, collected into standard vacuum tubes containing a 1/10th volume of trisodium citrate [Becton Dickenson USA, 0.109 mol/L in 4.5 mL blood (3.2%)] and immediately centrifuged at 2000 × g for 20 min at room temperature. Plasma was then frozen at -70 °C in aliquots of 0.5 mL until further analyses.
The study was performed in accordance with the Declaration of Helsinki and was approved by the Ethical Review Board in Stockholm, Sweden (reference numbers 2010/28-31/4 and 2012/1232-31/4). Oral and written informed consent was obtained from the participants.
Blood samples were drawn by direct venipuncture from an antecubital vein, collected into standard vacuum tubes containing a 1/10th volume of trisodium citrate [Becton Dickenson USA, 0.109 mol/L in 4.5 mL blood (3.2%)] and immediately centrifuged at 2000 × g for 20 min at room temperature. Plasma was then frozen at -70 °C in aliquots of 0.5 mL until further analyses.
The study was performed in accordance with the Declaration of Helsinki and was approved by the Ethical Review Board in Stockholm, Sweden (reference numbers 2010/28-31/4 and 2012/1232-31/4). Oral and written informed consent was obtained from the participants.
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