Informed written consent was obtained from all individuals, the study has been performed according to the Declaration of Helsinki, and the procedures have been approved by the Independent Local Ethics Committee of the University of Ulm, Ulm, Germany (no. 94/14). Whole blood from healthy volunteers was drawn into syringes containing 3.2% trisodium citrate (Sarstedt, Nümbrecht, Germany) as anticoagulant. Neutrophils were isolated by Ficoll-Paque gradient centrifugation (Amersham Biosciences, Solingen, Germany) followed by dextran sedimentation. After hypotonic lysis of residual erythrocytes, neutrophils were resuspended as indicated in Dulbecco’s phosphate-buffered saline (DPBS), Hank’s balanced salt solution with Ca2+/Mg2+ (HBSS) or RPMI 1640 (all Life Technologies, Darmstadt, Germany). For Amnis high-resolution digital imaging, neutrophils were isolated as described by Barnes et al. [21 (link)].
Trisodium citrate
Manufactured by Sarstedt
Sourced in Germany
Trisodium citrate is a chemical compound used as a buffer and anticoagulant in laboratory settings. It is a salt of citric acid with the chemical formula Na3C6H5O7. Trisodium citrate is commonly used to maintain a stable pH in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Kx 21, by Sysmex (1 mentions)
S monovette, by Sarstedt (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Kx 21n, by Sysmex (1 mentions)
Biocoll separating solution, by Merck Group (1 mentions)
10 protocols using trisodium citrate
1
Isolation and Characterization of Human Neutrophils
2
Blood Platelet Isolation and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was drawn by clean venipuncture and collected into vacutainer tubes containing
1/10 volume of 3.12% trisodium citrate as an anticoagulant (Sarstedt, Nümbrecht, Germany).
Platelet-rich plasma (PRP) was obtained by centrifugation of whole blood at 200 g for 20 min. Erythrocytes were collected from the bottom fraction and adjusted to the different numbers with plasma obtained after the second centrifugation to prepare PRP. To prepare plasma samples, PRP was centrifuged at 14000 g for 5 min. Platelet number was determined using a Coulter hematology analyzer. Volumes were equalized for all samples with autologous plasma (i.e., same volumes for all samples).
1/10 volume of 3.12% trisodium citrate as an anticoagulant (Sarstedt, Nümbrecht, Germany).
Platelet-rich plasma (PRP) was obtained by centrifugation of whole blood at 200 g for 20 min. Erythrocytes were collected from the bottom fraction and adjusted to the different numbers with plasma obtained after the second centrifugation to prepare PRP. To prepare plasma samples, PRP was centrifuged at 14000 g for 5 min. Platelet number was determined using a Coulter hematology analyzer. Volumes were equalized for all samples with autologous plasma (i.e., same volumes for all samples).
3
Isolation of Human Platelet Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The study conforms to the principles outlined in the “Declaration of Helsinki” (Cardiovascular Research 1997; 35: 2–3) for the use of human material. After approval from the local ethics committee of the University of Giessen (Giessen, Germany), peripheral blood was obtained from healthy human (male and female) volunteers (20–45 years old) who had not taken any drugs for at least 14 days. Blood samples were drawn into tubes containing trisodium citrate (Sarstedt, Germany). Whole blood was centrifuged at 110× g for 20 min at room temperature (RT) to obtain PRP. The platelet content was measured using an automatic haematology analyser Sysmex KX-21 (Sysmex, Germany). Platelet-poor plasma (PPP) was obtained by centrifugation of PRP at 14,000× g for 3 min. The platelet count in PRP was adjusted to (250–280) × 106/mL by diluting native PRP with the same donor’s PPP.
In order to obtain washed platelets, the PRP was centrifuged at 600× g for 20 min at RT. The platelet pellet was re-suspended in Tyrode’s buffer (pH 7.2) containing PGI2 (0.5 µM) and albumin (0.1%) and the suspension was re-centrifuged at 600× g for 10 min. Finally, the washed platelets were re-suspended in Tyrode’s buffer (pH 7.2) at the concentration of 3 × 108/mL. The suspended platelets showed a characteristic shimmering effect.
In order to obtain washed platelets, the PRP was centrifuged at 600× g for 20 min at RT. The platelet pellet was re-suspended in Tyrode’s buffer (pH 7.2) containing PGI2 (0.5 µM) and albumin (0.1%) and the suspension was re-centrifuged at 600× g for 10 min. Finally, the washed platelets were re-suspended in Tyrode’s buffer (pH 7.2) at the concentration of 3 × 108/mL. The suspended platelets showed a characteristic shimmering effect.
4
Prothrombin Time and INR Estimation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Patient samples for PT/INR estimation in this study were collected using 3.0 ml plastic plasma tubes (S-Monovettes) with sodium citrate, predosed as a 0.106 molar solution, which is equivalent to 3.2% trisodium citrate (Sarstedt AG & Co., Nümbrecht, Germany). PT was measured at 37°C on an ACL TOP 700 system using the HemosIL RecombiPlasTin 2G reagents on the basis of recombinant human tissue factor (Instrumentation Laboratory Company, Bedford, Massachusetts, USA).
The INR is the PT ratio of a test sample compared with a normal PT (derived from the log mean normal prothrombin time of 20 normal donors) corrected for the sensitivity of the thromboplastin used in the test. It is the value for the PT ratio that has been obtained using the first WHO reference thromboplastin with an international sensitivity index (ISI) of 1.0.
The INR value was calculated according to the following equation: The ISI web software and the certified plasmas of the HemosIL ISI Calibrate set (Instrumentation Laboratory Company) were used to establish a laboratory’s instrument/reagent-specific local ISI.
The INR is the PT ratio of a test sample compared with a normal PT (derived from the log mean normal prothrombin time of 20 normal donors) corrected for the sensitivity of the thromboplastin used in the test. It is the value for the PT ratio that has been obtained using the first WHO reference thromboplastin with an international sensitivity index (ISI) of 1.0.
The INR value was calculated according to the following equation: The ISI web software and the certified plasmas of the HemosIL ISI Calibrate set (Instrumentation Laboratory Company) were used to establish a laboratory’s instrument/reagent-specific local ISI.
5
Isolation of Human Neutrophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The investigations were approved by the Local Independent Ethics Committee of the University of Ulm (number 459/18; 94/14). After obtaining informed written consent from healthy human volunteers, blood was drawn into syringes containing 3.2% trisodium citrate (Sarstedt, Nürnbrecht, Germany). Neutrophils were isolated by Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation and subsequent dextran sedimentation followed by hypotonic lysis of remaining erythrocytes, as described previously (14 (link)–16 (link)). Polymorphonuclear granulocytes (mainly consisting of neutrophils) were adjusted to a concentration of 2 × 106 cells/ml using Hank's balanced salt solution with calcium and magnesium (HBSS, Thermo Fisher, Darmstadt, Germany), the pH of which was adjusted to 7.3, when not indicated otherwise.
6
Optimized Blood Coagulation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All included subjects provided blood samples with and without addition of CTI, which were measured with both CAT and TGA assays. For the blood sample without CTI, i.e., the standard sample, venous blood was collected with a 21-gauge needle using minimal suction with a light tourniquet from the antecubital vein into Sarstedt Monovette tubes containing 0.106 mol/L trisodium citrate. For the blood sample with CTI, i.e., the optimized sample, additional samples were drawn directly into 3mL Sarstedt tubes containing 0.35mL of citrate/CTI; a mix of 0.3mL 0.106mol/L trisodium citrate (Sarstedt, Leicester, UK) and 0.05mL CTI (1.1mg/mL) resulting in a final CTI concentration of 18.3 μg/mL in whole blood. Of both standard and optimized blood samples platelet poor plasma was obtained by centrifugation one time for 10 minutes at 3000g at room temperature and stored at -70°C until it was analysed.
7
Isolation of Human Platelets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Our studies with human platelets and the consent procedure were approved by the local Ethics Committee of the University of Wuerzburg (approval number: 101/15). All participants provided their written informed consent. The study was performed according to our institutional guidelines and to the Declaration of Helsinki.
Peripheral venous blood (PB) from informed healthy voluntary donors was collected in polypropylene tubes containing 3.2% citrate buffer (106 mM trisodium citrate, Sarstedt, Nuembrecht, Germany). To prepare washed platelets (WP), 3-mM EGTA was added to PB prior to centrifugation to prevent platelet activation. Platelet-rich plasma (PRP) was obtained by centrifugation at 280 × g for 5 minutes. Then, 75-nM PGE1 was added to PRP, and platelets were pelleted at 430 × g for 7 minutes. The pelleted platelets were then washed once in chloride/glucose/sodium (CGS) buffer (120-mM sodium chloride, 12.9-mM trisodium citrate, 30-mM D-glucose, and pH = 6.5) and resuspended in Tyrode's salt solution to a final concentration of 3 × 10
8platelets/mL.
Peripheral venous blood (PB) from informed healthy voluntary donors was collected in polypropylene tubes containing 3.2% citrate buffer (106 mM trisodium citrate, Sarstedt, Nuembrecht, Germany). To prepare washed platelets (WP), 3-mM EGTA was added to PB prior to centrifugation to prevent platelet activation. Platelet-rich plasma (PRP) was obtained by centrifugation at 280 × g for 5 minutes. Then, 75-nM PGE1 was added to PRP, and platelets were pelleted at 430 × g for 7 minutes. The pelleted platelets were then washed once in chloride/glucose/sodium (CGS) buffer (120-mM sodium chloride, 12.9-mM trisodium citrate, 30-mM D-glucose, and pH = 6.5) and resuspended in Tyrode's salt solution to a final concentration of 3 × 10
8platelets/mL.
8
Platelet Preservation and Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Our studies with human platelets and the consent procedure were approved by our local ethics committee of the University of Wuerzburg (approval number 101/15). The participants provided their written informed consent to participate in this study. The study was performed according to our institutional guidelines and to the Declaration of Helsinki.
Venous whole blood samples were obtained from informed healthy voluntary donors without any medication intake. Peripheral blood was collected in polypropylene tubes containing 3.2% citrate buffer (106 mM trisodium citrate, Sarstedt, Nuembrecht, Germany).
Platelet-rich-plasma (PRP) was prepared by centrifugation of whole blood at 280 × g for 5 min (min) as described10 (link).
Blood cell count was performed with the hematology analyzer Sysmex KX-21 N (Sysmex Europe GmbH, Norderstedt, Germany). PRP was divided into three parts (each 4.5 mL PRP in a polypropylene tube), which were stored at RT (20–24 °C) for 2 h (control), stored for 1 h at CT (2–6 °C) or stored for 2 h at CT. Before consecutive analysis, platelets were left quiescent at RT for 30 min.
Venous whole blood samples were obtained from informed healthy voluntary donors without any medication intake. Peripheral blood was collected in polypropylene tubes containing 3.2% citrate buffer (106 mM trisodium citrate, Sarstedt, Nuembrecht, Germany).
Platelet-rich-plasma (PRP) was prepared by centrifugation of whole blood at 280 × g for 5 min (min) as described10 (link).
Blood cell count was performed with the hematology analyzer Sysmex KX-21 N (Sysmex Europe GmbH, Norderstedt, Germany). PRP was divided into three parts (each 4.5 mL PRP in a polypropylene tube), which were stored at RT (20–24 °C) for 2 h (control), stored for 1 h at CT (2–6 °C) or stored for 2 h at CT. Before consecutive analysis, platelets were left quiescent at RT for 30 min.
9
PRP Preparation and PPP Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Laboratory tests were performed on platelet‐rich plasma (PRP) for patients from centers 1 and 2. Blood was drawn into S‐Monovette tubes, mixed therein with a 1:10 volume of 0.106 mol/L tri‐sodium citrate (Sarstedt, Mamay, France), and centrifuged at 140 g for 10 minutes at 20°C. Platelet count was not adjusted. PRP was used within 2 hours after blood collection. To obtain platelet‐poor plasma (PPP), blood was centrifuged twice at 2500 g for 15 minutes. PPP was stored at −80°C and thawed for 5 minutes in a water bath at 37°C before TG assay.
10
PBMC Isolation and Biobanking from ACC Patients
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMC) were obtained from patients suffering from ACC (n = 11) and from normal controls (NC; n = 11), matched by age (± 5 years), who signed an informed consent form approved by the local ethical committee of the Ulm University (#255/14). At the time of blood withdrawal, eight patients suffered from an active disease (AD), five patients were within their first year after surgery (SRG), six patients had undergone radiotherapy (RT) within 12 months before blood withdrawal and five patients had a recurrent tumor or distant metastasis (R/M) within 1 month before blood withdrawal. Tumors of patients with surgery as primary treatment had negative margins, except for case #7, which had minimal residual disease before undergoing adjuvant RT. A total of 24 samples were collected of the patients during this study. Blood (50 ml) was collected in S-monovettes prefilled with trisodium citrate (Sarstedt) and centrifuged on Biocoll Separating Solution (Merck). PBMC were recovered, washed twice in PBS and stored for further experiments in a freezing medium containing FBS and DMSO in liquid nitrogen. The patients presented with different tumor sites and blood was drawn at different time points during treatment and course of disease. Detailed patient characteristics are listed in Table 1.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!