A total of three venous blood samples from healthy individuals were collected at the Department of Clinical Chemistry at Hannover Medical School. Informed consent was obtained from all donors before blood sampling. The experiments were carried out in accordance with the relevant guidelines and regulations and approved by the Hannover Medical School ethics committee in accordance with the Declaration of Helsinki (No. 388-2008, 02.12.2008). Blood sampling was performed using commercially available heparin- (i.e., HMWH) containing monovette tubes (Sarstedt, Nümbrecht, Germany). Blood samples were immediately centrifuged at 1900× g for 10 min and after removal of the supernatant, plasma samples were aliquoted and stored at −80 °C until assay.
Monovette tube
Manufactured by Sarstedt
Sourced in Germany, United Kingdom
The Monovette tubes are a system of blood collection tubes manufactured by Sarstedt. The tubes are designed for the safe and convenient collection of blood samples. The Monovette tubes come in a variety of sizes and configurations to accommodate different blood collection needs.
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Lab products found in correlation
Vacutainer vacuum tubes, by BD (2 mentions)
Pm100, by Mettler Toledo (1 mentions)
Safety multifly needle 21g, by Sarstedt (1 mentions)
Cacl2, by Siemens (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Pancoll, by PAN Biotech (1 mentions)
Automated analyzer, by Abbott (1 mentions)
Cell dyn 3700, by Abbott (1 mentions)
Modular swa, by Roche (1 mentions)
25 protocols using monovette tube
1
Healthy Individual Plasma Collection
2
Monocyte-to-HDL Cholesterol Ratio
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Peripheral venous blood samples were obtained from the patients on their admission. An automated blood cell counter (Beckman Coulter analyzer, California, USA) was used for measuring complete blood count (CBC) parameters, and CBC samples were collected in ethylenediaminetetraacetic acid (EDTA)-anticoagulated Monovette ® tubes (Sarstedt). Monocyte count was calculated from the CBC analysis. The biochemical parameters, such as total cholesterol, HDL-C, low-density lipoprotein cholesterol (LDL-C), and triglyceride levels, were also measured. The MHR was calculated as the ratio of monocytes to HDL-C levels.
3
Standardized Blood Culture Protocol
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Internal hospital standard operating procedures specify that nursing staff should fill two Monovette® tubes (Sarstedt, Nuembrecht, Germany) with 8–10 mL of blood by single sampling strategy taken under aseptic conditions without air supply and transfer the blood from each tube to an aerobic and an anaerobic BC. BC bottles are immediately sent to the laboratory for further processing.
The BC systems used are the BacT/ALERT FA FAN ® Aerobic and BacT/ALERT FN FAN® Anaerobic (bioMérieux, Roissy CDG, France) bottles. Aerobic and anaerobic bottles contain 30 mL and 40 mL of nutrient agar, respectively. BC bottles were incubated in VirtuO BacT/ALERT devices (bioMérieux) until positive or for a maximum of six days. The time to positivity was documented. Positive bottles were analyzed with subsequent standard microbiological methods [11 (link)].
Each BC was individually weighed to 0.1 g accuracy using an electronic high-precision scale (PM100, Mettler-Toledo, Greifensee, Switzerland). The blood volume (in mL) was calculated according to Henning et. al [6 ]. Briefly, estimated volume = (weight of bottle filled with blood [g]– mean weight of standard bottles without blood [g]) / density of blood (1.055 [g/mL]). We determined the median weight and IQR of 20 aerobic and anaerobic BCs bottles without blood, which was 61.6 g (IQR 61.5–61.6) and 72.15 g (IQR 71.7–72.3), respectively.
The BC systems used are the BacT/ALERT FA FAN ® Aerobic and BacT/ALERT FN FAN® Anaerobic (bioMérieux, Roissy CDG, France) bottles. Aerobic and anaerobic bottles contain 30 mL and 40 mL of nutrient agar, respectively. BC bottles were incubated in VirtuO BacT/ALERT devices (bioMérieux) until positive or for a maximum of six days. The time to positivity was documented. Positive bottles were analyzed with subsequent standard microbiological methods [11 (link)].
Each BC was individually weighed to 0.1 g accuracy using an electronic high-precision scale (PM100, Mettler-Toledo, Greifensee, Switzerland). The blood volume (in mL) was calculated according to Henning et. al [6 ]. Briefly, estimated volume = (weight of bottle filled with blood [g]– mean weight of standard bottles without blood [g]) / density of blood (1.055 [g/mL]). We determined the median weight and IQR of 20 aerobic and anaerobic BCs bottles without blood, which was 61.6 g (IQR 61.5–61.6) and 72.15 g (IQR 71.7–72.3), respectively.
4
Appendicular Fluid Sampling Protocol
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After appendectomy, a gauge was inserted in the caecal side of the lumen of the appendix and 3 mL of saline 0.9% was administered and collected, and re-administered and re-collected 3 additional times.
Previous training was carried out for appendicular lavage so that the process was standardized and data collection uniformised. A tutorial was presented to all the surgeons that performed an appendectomy. The appendicular fluid samples were collected with Sarstedt Monovette tubes and centrifuged. A total of 1 mL of the supernatant was extracted and stored at −20 °C [5 (link)].
Previous training was carried out for appendicular lavage so that the process was standardized and data collection uniformised. A tutorial was presented to all the surgeons that performed an appendectomy. The appendicular fluid samples were collected with Sarstedt Monovette tubes and centrifuged. A total of 1 mL of the supernatant was extracted and stored at −20 °C [5 (link)].
5
Plasma Collection and Storage
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Blood was drawn from all patients at diagnosis and every three months during follow-up. Blood was collected in Monovette tubes (Sarstedt, North Rhine-Westphalia, Germany) containing 0.109 M trisodium citrate and then centrifuged at 2600× g for 20 min at 4 °C. Plasma was collected ensuring the absence of platelet and leukocyte contamination, by only taking the 2/3 of upper plasma, and stored in aliquots at −80 °C until used.
6
Thrombin Generation Assay Protocol
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Blood was collected in Monovette tubes (0.106 M citrate, 5 mL, Sarstedt, Nümbrecht, Germany) with and without manually prefilled CTI (18.3 μg/mL final concentration). The venipuncture was performed with a Safety-Multifly®-Needle 21G (Sarstedt, Nümbrecht, Germany), and the first tube was discarded. After written informed consent, eight healthy volunteers donated blood to four tubes with CTI and four tubes without CTI. The tubes were centrifuged (2,000 g, 15 min, RT) and pooled into two separate plasmas (+/- CTI) which were subjected to another centrifugation at 2,000 g, 15 min, RT. The PNP (+/- CTI) was aliquoted and stored at -80°C. At the day of use, the PNP (+/- CTI) was thawed at RT, and incubated (15 min at 37°) with anti-human TFPI abs (100 μg/mL final conc.) or an equal volume of TBSA before addition to the wells. The concentration of anti-TFPI abs was chosen based on initial in vitro thrombin generation experiments, where 100 μg/mL of anti-TFPI abs gave the highest sensitivity for detection of MVs obtained from whole blood stimulated with Neisseria meningitidis bacteria.
7
PON1 Genotyping and Activity
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Blood probes for PON1 genotyping and serum PON1 activity were taken before a midweek dialysis session when HD patients had collected blood for routine periodical laboratory testing. Monovette tubes (SARSTEDT, Nümbrecht, Germany) were used for venous blood sampling. Tubes containing the EDTA anticoagulant were applied for DNA analyses and blood morphology. If serum was needed, blood samples were drawn to Monovette tubes allowing the blood clotting (no anticoagulant). PON1 activity, cholesterols, TG, creatinine, urea, CRP, albumin, calcium, phosphorus, alkaline phosphatase (ALP), and parathyroid hormone were determined in serum.
8
Chitosan-Based Anticoagulant Formulation
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Chitosan 95/100 [degree of deacetylation >92.6%, viscosity ≤71–150 mPas, MW 100–250 kDa (SEC)] was obtained from Heppe Medical Chitosan GmbH (Halle, Germany). NaVS [tech., 25% (aq)] was purchased from Alfa Aesar GmbH & Co. KG (Karlsruhe, Germany) and sodium 2-bromoethanesulfonate (NaBES, 98%) from Sigma-Aldrich Co. (St Louis, MO, USA). Dialysis membranes (Spectra/Por® Biotech CE, molecular weight cutoff: 100–500 Da) were received from Spectrum Laboratories, Inc. (Rancho Dominquez, CA, USA). Silver nitrate (99%; Merck, Darmstadt, Germany) was used without further purification. Human blood plasma was used for all coagulation assays and was received from a single donor to ensure comparability of consecutive tests. Citrated blood samples were obtained by clean venipuncture using 19-gauge butterfly needles and Monovette tubes (Sarstedt, Nümbrecht, Germany) containing 1/9 vol of 0.106 M sodium citrate. Plasma was prepared by double centrifugation [1,500 ×g, each for 10 minutes, at room temperature (RT)] and was stored in polypropylene tubes at −70°C until use. CaCl2 (medical grade) was received from Siemens Healthcare Diagnostics Products (Marburg, Germany). All other chemicals were utilized in analytical grade. Ultrapure water (MilliQ, Ω≤18.2 MΩ cm) was used for all applications.
9
Cryopreservation of Human PBMCs
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ETDA-anticoagulated venous blood samples were collected in Monovette tubes (Sarstedt, Nuembrecht, Germany). PBMCs were isolated by density gradient centrifugation using Pancoll (PAN-Biotech, Aidenbach, Germany) and washed with phosphate-buffered saline (PBS, PAN-Biotech). The cells were cryopreserved at −80 °C. Thawing of PBMCs was performed in complete medium (RPMI1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES; Fisher Scientific GmbH, Schwerte, Germany).
10
Intracardiac Blood Withdrawal for Liver and Kidney Analysis
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Following anesthesia prior to sacrifice, blood was withdrawn intracardially with a needle and syringe, transferred to Monovette® tubes (Sarstedt, Nümbrecht, Germany) and analyzed the same day for testing standard liver and kidney functions by the department of laboratory medicine (Klinikum rechts der Isar, Munich, Germany).
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