Elbow venous blood was collected in a fasting state from 8 AM to 9 AM. For menstruating women, the blood was sampled on the 3rd–5th day after menstruation. For women whose menstruation had stopped for ≥6 months, the blood was sampled on any day according to convenience. The blood samples were divided as follows: 5 mL in a procoagulant tube, 3 mL treated with anticoagulant (EDTA-K2; Sarstedt, Niimbrecht, Germany), and 1.5 mg/mL untreated for further use.
K2 edta
Manufactured by Sarstedt
Sourced in Germany
K2 EDTA is a laboratory product used as an anticoagulant in blood collection and analysis. It acts as a chelating agent, binding to calcium ions to prevent blood clotting. This preserves the sample for further testing and evaluation.
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6 protocols using k2 edta
1
Blood Sample Collection and Preparation
2
Comprehensive Metabolic Profiling of Mice
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At 12 h 00 am, after 4 h of fast, mice were anesthetized under isoflurane (5 L/min, 2–3%) and blood was collected by cardiac puncture into tubes containing EDTA K2 (Sarstedt, Marnay, France). Organs were rapidly excised and snap frozen in liquid nitrogen before being stored at −80 °C. Aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) were determined by photometry using standard clinical biochemical procedures (Roche Diagnostics, Meylan, France). Fasting plasma total cholesterol (TC), triglycerides (TG) and non-esterified fatty acids (NEFA) were performed using enzymatic assay (DiaSys, Grabels, France). Isolation of lipoproteins was performed by fast protein liquid chromatography (ÄKTAFPLC) (GE Healthcare, Buc, France) using 200µL of plasma [16 (link)]. Plasma apolipoproteins (ApoA-I, ApoB100, ApoC-II, ApoC-III, and ApoE) were measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) as previously described [17 (link)] with slight modifications due to specific mouse isoforms (Table S3 ). Gallbladder bile acids (BA) were further analyzed by LC-MS/MS as recently published [18 (link)]. Thiobarbituric acid reactive substance (TBARS) content in liver were quantified using the fluorimetric procedure of Yagi [19 (link)].
3
Testosterone Levels in Children
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Venous blood samples were drawn from all 60 children into sterile polypropylene tubes containing K2 EDTA (Sarstedt, Nümbrecht, Germany) using standardized procedures the same month of the year from 8:00 to 10:00 a.m. in respect of the circadian [48 (link),49 (link)] and infradian [50 (link),51 (link)] rhythm of testosterone fluctuations from all children at the Pediatric Department of Children Faculty Hospital CU in Bratislava. Whole blood samples were centrifuged for 5 min at 2000 g immediately after collection. Plasma aliquots were stored at −20°C for not longer than one month. On the day of testing, frozen samples were brought to room temperature and pipetted on to a testing plate. The ELISA assay using a commercial Testosterone ELISA kit was used according to manufacturer's instructions (DRG Instruments GmbH, Marburg, Germany). The intra-assay coefficient of variation was lower than 5% and the inter-assay coefficient of variation was 10%in every measurement. Unfortunately, plasmatic testosterone levels were measured only in 40 boys due to lack of blood sample volumes.
4
Newborn Sickle Cell Screening Protocol
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For initial development of the paper-based newborn SCD screening test, blood samples were collected from SCD (HbSS) patients at the Texas Children’s Hematology Center and from healthy volunteers (HbAA) into Vacutainer vials (K2EDTA, BD Diagnostics, USA) using standard venipuncture technique. Samples were stored at 4 °C until use. Artificially reconstituted samples with a range of HbS levels were created by mixing ABO/Rh-matched, equal-hematocrit HbAA and HbSS blood samples at various ratios.
For test validation in Cabinda, blood samples were collected from newborns by heel-stick onto blood collection cards (Whatman 903 Protein Saver Card, GE Healthcare, USA) and into capillary blood collection tubes (Microvette, K2EDTA, Sarstedt AG & Co, Germany). Eluted dried blood spot samples were tested with isoelectric focusing (IEF) following existing standard operating procedures7 (link). Liquid blood samples were refrigerated and used to perform the paper-based test within 7 days of collection. For all patients, the paper-based test was completed before IEF analysis. Local health workers interpreted the results of the paper-based test visually, using reference images of HbS-positive and HbS-negative bloodstains.
For test validation in Cabinda, blood samples were collected from newborns by heel-stick onto blood collection cards (Whatman 903 Protein Saver Card, GE Healthcare, USA) and into capillary blood collection tubes (Microvette, K2EDTA, Sarstedt AG & Co, Germany). Eluted dried blood spot samples were tested with isoelectric focusing (IEF) following existing standard operating procedures7 (link). Liquid blood samples were refrigerated and used to perform the paper-based test within 7 days of collection. For all patients, the paper-based test was completed before IEF analysis. Local health workers interpreted the results of the paper-based test visually, using reference images of HbS-positive and HbS-negative bloodstains.
5
Plasma Biomarker Collection in Mice
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Blood was collected at the start of the study and before first dosage to assess baseline conditions. After 3 months of dosing, blood was collected before the mice were sacrificed for the LTP measurement. Blood was collected using Microvette CB 300 tubes with K2EDTA (Sarstedt; cat 16.444). Conscious mice were restrained either manually or using a restraint tube and 150 μl sample was collected from the lateral saphenous vein. Tubes were stored at 4 °C for no more than 1 h. Samples were centrifuged in Microvette tubes down at 2000 g at 4 °C for 15 min. 20 μl of plasma was transferred to labelled tubes and color and letter-coded to treatment conditions. Samples were frozen in liquid N2 and then stored at −80 °C till analysis of AD biomarker profile.
6
Pediatric Hormone Assessment Protocol
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Venous blood samples were drawn into sterile polypropylene tubes containing K2 EDTA (Sarstedt, Nümbrecht, Germany) using standardized procedures the same month of a year from 8:00 to 10:00 a.m. from all children at the Pediatric Department of Children Faculty Hospital CU in Bratislava. Whole blood samples were centrifuged for 5 min at 2000 g immediately after collection. Plasma aliquots were stored at -20 °C for not longer than one month. On the day of testing, frozen samples were brought to room temperature and pipetted on to a testing plate. The ELISA assay using a commercial Testosterone ELISA kit and an Estradiol ELISA kit were used according to manufacturer's instructions (DRG Instruments GmbH, Marburg, Germany). The intra-assay coefficient of variation was 3.3 % and the inter-assay coefficient of variation was 6.2 %.
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