Blood samples were collected by venepuncture from the median cubital vein into two 5 mL sodium citrate vacutainers (Becton Dickinson). PMNs were isolated using 1-Step Polymorphs (Accurate) according to the manufacturer’s instructions. Residual erythrocytes were lysed with 1 mL of distilled water and recovered PMNs were washed in Hank’s balanced salt solution without calcium and magnesium (HBSS-/-, Gibco) and centrifuged at 1500 RCF for five minutes. The supernatant was discarded and the PMN pellet was resuspended in 1 mL phosphate buffered saline without calcium and magnesium (PBS-/-, Sigma-Aldrich). Recovered blood PMNs were quantified using a Z2 Cell and Particle Counter (Beckman Coulter) and demonstrated greater than 95% cell viability and 98% purity as assessed by a trypan blue exclusion test and Kwik-Diff staining (Thermo Fisher Scientific), respectively.
Sodium citrate vacutainers
Manufactured by BD
Sourced in United States
Sodium citrate vacutainers are a type of blood collection tube used for the collection and storage of blood samples. They contain a sodium citrate anticoagulant solution that helps prevent blood clotting during the collection process.
Automatically generated - may contain errors
Lab products found in correlation
Sodium citrate vacutainer tubes, by BD (2 mentions)
Z2 cell and particle counter, by Beckman Coulter (1 mentions)
Kwik diff staining, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s media, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Xe 2100 automated hematology system, by Sysmex (1 mentions)
K2edta, by BD (1 mentions)
12 protocols using sodium citrate vacutainers
1
Isolation and Characterization of Human PMNs
2
Rabbit Blood Sampling for Ex Vivo Analysis
3
Microfluidic Platelet Adhesion Assay
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Bovine serum albumin (BSA) (A9418), 3,3’-dihexyloxacarbocyanine iodide (DiOC6) (D273), glutaraldehyde (340855) heparin salt (H3393), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (H3375), poloxamer 407 (Pluronics® F-127, P2443) were from Sigma–Aldrich (St Louis, MO, USA). 3.2% sodium citrate vacutainers (369714) and 21-gauge Vacutainer® 21 Safety-Lok blood collection sets (367281) were from Becton Dickson (Frankwood Lakes, NJ, USA). 500 µL and 100 uL glass luer lock syringes were from Hamilton (Reno, NV, USA). Tridecafluoro-1,1,2,2-tetrahydrooctyltrichlorosilane (FOTS) (SIT8174.0) was from Gelest (Morrisville, PA, USA). Polydimethylsiloxane and crosslinker were obtained from Krayden (Denver, CO, USA). Collagen related peptides (CRP-XL) [GCO(GPO)10GCOG-amide], 5(6)-carboxyfluorescein (FAM) labeled CRP-XL, GFOGER [GPC(GPP)5GFOGER(GPP)5GPC-amide], and VWF-III [GPCGPP)5GPRGQOGVMGFO(GPP)5GPC-amide] were obtained from Cambcol Laboratories (Cambridgeshire, UK). Glass slides (2” x 3“) were obtained from Fisher Scientific (Lenexa, KS, USA). Pierce Quantitative Fluorometric Peptide Assay (23290) and Texas Red dye (T20175) were from Thermo Fischer (Denver, CO, USA). Phosphate-buffered saline (PBS) was prepared to 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and pH 7.4.
4
Extracellular Vesicle Isolation from Biofluids
5
Platelet Function in Surgical Patients
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Age and sex were reported at baseline. Height and weight were obtained from medical notes. BMI was derived from weight and height (kg/m 2 ). Smoking was reported as a categorical variable (0 = never smoker, 1 = ex-smoker for >5 years, 2 = ex-smoker for 1-5 years, 3 = ex-smoker for 30 days-1 year, 4 = current smoker). Platelet variables (PLT, MPV, IPF, and IPC) were measured using the Sysmex XE-2100 Automated Hematology System (Oxford, UK). Blood samples were taken pre-operatively into 3.2% sodium citrate vacutainers (BD Biosciences, Milton Keynes, UK). Platelet aggregation was measured using Multiplate multiple electrode aggregometry (MEA) (Roche, Rotkreuz, Switzerland), which detects change in electrical impedance when platelets aggregate on metal electrodes. Aggregation was determined by the area under the curve (AUC) in response to platelet agonists, including adrenaline (100 mg/mL), thrombin receptor activator peptide 6 test (TRAP-test), ADP-test, and ASPItest. Using of a combination of agonists enables evaluation of platelet activation via different pathways: adrenaline acts at the α2 adrenergic receptor, TRAP-6 acts at the protease-activated receptor-1 (PAR-1), ADP acts at the P2Y 12 receptor and arachidonic acid at the Thromboxane A2 (TxA2) receptor.
6
Coagulation Assay Protocol: PT and aPTT
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Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured on the KC4 delta coagulometer (TCoag, Wicklow, Ireland) as previously described. 14 Briefly, arterial blood collected in 3.2% sodium citrate vacutainers (BD, North Ryde, NSW, Australia) was centrifuged at 1500 g at room temperature, and plasma stored at À80 C until analysis. PT and aPTT were determined using STA-Neoplastine Õ Cl Plus (Lot: 250461) and TriniCLOT aPTT HS reagent (Lot: G203641) (Diagnostica Stago, Donacaster, VIC, Australia). Measurements were performed in triplicate, and assays were terminated after 5 min.
7
Isolation of Blood Components for Analysis
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All human studies were approved by the University of Alabama at Birmingham Institutional Review Board for Human Use with all subjects providing written consent. Peripheral blood was collected from healthy volunteers by venipuncture into serum, heparin sulfate, K2EDTA, and sodium citrate vacutainers (BD Biosciences) during a single draw. Samples were kept at room temperature until blood in the serum tube was coagulated. Afterwards, samples were centrifuged at 400xg for 10 minutes to separate blood cells from serum or plasma. Samples were either directly aliquoted and stored at −80°C or given an additional round of centrifugation at 3000xg for 10 minutes to separate platelets from serum or plasma. All serum and plasma sample values depicted in figures were free of platelets. All experiments had 6 samples per group except for the experiments depicted in Figures 2 and 4 .
8
Platelet-Poor Plasma Preparation
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Peripheral blood samples were collected in two 3mL sodium citrate vacutainers (BD Biosciences, India). One tube was used for ROTEM, and the second tube was used to prepare platelet-poor plasma (PPP). Plasma was separated by centrifugation at 2,000 g (3,500 rpm) for 15 min to prepare the PPP.
9
Coagulation Experiments with Healthy and Diseased Blood
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Blood samples were collected after informed, written consent as per ethical guidelines of Institutional Review Board of Partners Healthcare, Harvard Medical School and Beth Israel Deaconess Medical Center. Healthy donors: blood was collected from non-smoking healthy volunteers in a standard 6 ml no-additive blood vacutainer (BD) and 1000 U ml−1 unfractionated heparin was immediately added to a required concentration. Coagulation experiments were initiated within ∼15 min after the blood draw. For experiments where heparin concentration was <0.25 IU ml−1, blood was first drawn in 3.2% 5 ml sodium citrate vacutainers (BD) or purchased (Research Blood Components, Cambridge, MA). Patients: subjects who were taking aspirin and plavix (clopidogrel) were selected from among patients seen in the cardiac catheterization laboratory at Beth Israel Deaconess Medical Center, Boston, MA. Whole blood from patients with HPS was drawn using standard phlebotomy techniques and stored in 4.5 ml citrated (3.2% sodium citrate) tubes at room temperature. Citrated blood was used within 5 h of blood draw. The coagulation activity of these samples was restored by adding 100 μl ml−1 of 100 mM calcium chloride/75 mM magnesium chloride solution.
10
Platelet-Free Plasma Isolation for CLL Study
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Blood was collected from three healthy donors (25, 32 and 45 years of age) using a 21-gauge needle. The first 3 mL of blood was discarded and approximately 8 mL was transferred within sodium citrate Vacutainers (BD Biosciences Inc.). Platelet-free plasma (PFP) was isolated within 1 hour post-collection by double centrifugation at 2,500 Â g for 15 minutes (room temperature [RT], no brake) according to the International Society on Thrombosis and Haemostasis (ISTH) guidelines. 34 Plasmas were aliquoted and stored at -80°C. Chronic lymphocytic leukaemia (CLL) plasma samples consisting of both low radioactive iodine (RAI) risk (stage 0-1) and high RAI risk (stage 3-4) were obtained from the Division of Hematology (Dr. Neil Kay) under Institutional Review Board #1827. PFPs were prepared as above and stored at -80°C until further use.
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