Peripheral blood was obtained from 5 adult male healthy donors recruited to the IRCCS-Humanitas Research Hospital. The study protocol was approved by the ethical committee of Humanitas Clinical and Research Center (Prot. Nr 520/18, approved on 9/2018). All participants gave written informed consent. Samples were collected in EDTA-coated tubes (BD Vacutainer K2E), and peripheral blood mononuclear cells (PBMCs) were isolated by Lympholyte® cell separation density gradient solution (Cederlane). Any residual erythrocytes were removed via ammonium-chloride-potassium (ACK) Lysing Buffer (Lonza) treatment for 60 sec at room temperature (RT).
Vacutainer k2e
Manufactured by BD
Sourced in United Kingdom, United States
The BD Vacutainer K2E is a blood collection tube designed to collect and store blood samples. It contains the anticoagulant K2EDTA, which helps prevent the blood from clotting during the collection and transportation process.
Automatically generated - may contain errors
Lab products found in correlation
Microtube, by Corning (5 mentions)
Vacutainer 9nc, by BD (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Vacutainer sst 2 advance, by BD (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Vacutainer, by BD (2 mentions)
Ssttmii advance vacutainer, by BD (2 mentions)
Lymphoprep, by STEMCELL (2 mentions)
Leptin, by Elabscience (1 mentions)
Glucagon, by Elabscience (1 mentions)
Adiponectin, by Elabscience (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Ultra 2 fs dna library prep kit, by Illumina (1 mentions)
Qiaamp dna micro kit, by Qiagen (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Ficoll, by GE Healthcare (1 mentions)
Facs lysing solution, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (1 mentions)
Taqman rnase p assay, by Thermo Fisher Scientific (1 mentions)
46 protocols using vacutainer k2e
1
Isolation of Human PBMCs
2
Venous Blood Sampling for Biomarker Analysis in MI
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Venous blood samples were collected in ice-chilled tubes coated with ethylenediaminetetraacetic acid (EDTA; BD Vacutainer K2E; BD Diagnostics, NJ, USA) on the day before MI and day 1, 7, and 30 following MI. The plasma was separated by centrifugation at 3,000 rpm for 10 min at 4 °C and stored at −80 °C until assayed. Norepinephrine (NE) and N-terminal pro-B-type-natriuretic peptide (NT-proBNP) concentrations were determined with a radioimmunoassay kit from the American Laboratory Products Company (Salem, NH, USA) according to the manufacturer’s procedure.
3
Blood Lipid and Hormone Profiling in Experimental Animal Study
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Triglyceride, total cholesterol and high-density lipoprotein (HDL) cholesterol levels were measured in the serum samples using a clinical chemistry analyzer (Olympus AU480, Beckman Coulter, Brea, CA, USA). Serum samples were obtained by centrifugation (1000× g, 15 min) of blood samples taken from the jugular vein in plastic tubes with clot activator (BD Microtainer® SST II).
The concentrations of insulin, leptin, adiponectin (Demeditec Diagnostics, Kiel, Germany) and glucagon (Elabscience, Wuhan, China) were determined in the plasma samples using a commercially available enzyme-linked immunosorbent assay (ELISA) with a sensitivity of 0.1 ng/mL (insulin), 0.081 ng/mL (adiponectin), <0.25 µg/mL (leptin) and 37.5 pg/mL (glucagon). Plasma samples were prepared by centrifugation (1500× g, 10 min) of blood samples taken from the jugular vein in plastic tubes containing EDTA salts (BD Vacutainer™ K2E), and were stored in aliquots at −80 °C until analyses were performed.
Glucose levels were determined in blood samples taken from an incision at the tip of the tail. Measurements were performed with a glucometer (iDIA, Hof, Germany) using the glucose oxidase method.
The Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated using the following formula: fasting plasma insulin (mIU/L) * fasting blood glucose (mmol/L)/22.5.
The concentrations of insulin, leptin, adiponectin (Demeditec Diagnostics, Kiel, Germany) and glucagon (Elabscience, Wuhan, China) were determined in the plasma samples using a commercially available enzyme-linked immunosorbent assay (ELISA) with a sensitivity of 0.1 ng/mL (insulin), 0.081 ng/mL (adiponectin), <0.25 µg/mL (leptin) and 37.5 pg/mL (glucagon). Plasma samples were prepared by centrifugation (1500× g, 10 min) of blood samples taken from the jugular vein in plastic tubes containing EDTA salts (BD Vacutainer™ K2E), and were stored in aliquots at −80 °C until analyses were performed.
Glucose levels were determined in blood samples taken from an incision at the tip of the tail. Measurements were performed with a glucometer (iDIA, Hof, Germany) using the glucose oxidase method.
The Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated using the following formula: fasting plasma insulin (mIU/L) * fasting blood glucose (mmol/L)/22.5.
4
Healthy Donor Plasma Extraction and HLA Typing
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Plasma was obtained by withdrawing blood from eight healthy donors (specified in supplemental Table S1 ) in EDTA tubes (BD Vacutainer K2E; REF 367525). The tubes were inverted three times and centrifuged at 2000g for 20 min at 4 °C. The plasma was separated, aliquoted, snap-frozen, and stored at −80 °C.
To determine the HLA types of healthy donors, genomic DNA was extracted from buccal swabs. Cotton swabs were used to obtain oral mucosa samples, placed in protease buffer (30 mM Tris-HCl, pH 8, 0.5% Tween-20, 0.5% IGEPAL CA-630), and proteins were digested with 100 μg proteinase K per sample for 12 min at 50 °C in a thermal shaker. The enzyme was inactivated at 75 °C for 30 min and genomic DNA was purified using the QIAamp DNA Micro Kit (Qiagen). HLA class I (HLA-A, HLA-B, HLA-C) and class II (DRB1, DQB1, DPB1) loci were amplified using the NGSgo-MX6-1 kit (GenDx), and multiplexed sequencing libraries were prepared using the Ultra II FS DNA Library Prep Kit for Illumina (NEB). Libraries were sequenced on a NovaSeq 6000 system (Illumina) in 150 bp paired-end mode and the genotype data were analyzed using the NGSengine software (GenDx). Blood was sampled from healthy donors, who provided written informed consent, with prior approval of the ethics committee of the Max Planck Society in accordance with the Declaration of Helsinki principles.
To determine the HLA types of healthy donors, genomic DNA was extracted from buccal swabs. Cotton swabs were used to obtain oral mucosa samples, placed in protease buffer (30 mM Tris-HCl, pH 8, 0.5% Tween-20, 0.5% IGEPAL CA-630), and proteins were digested with 100 μg proteinase K per sample for 12 min at 50 °C in a thermal shaker. The enzyme was inactivated at 75 °C for 30 min and genomic DNA was purified using the QIAamp DNA Micro Kit (Qiagen). HLA class I (HLA-A, HLA-B, HLA-C) and class II (DRB1, DQB1, DPB1) loci were amplified using the NGSgo-MX6-1 kit (GenDx), and multiplexed sequencing libraries were prepared using the Ultra II FS DNA Library Prep Kit for Illumina (NEB). Libraries were sequenced on a NovaSeq 6000 system (Illumina) in 150 bp paired-end mode and the genotype data were analyzed using the NGSengine software (GenDx). Blood was sampled from healthy donors, who provided written informed consent, with prior approval of the ethics committee of the Max Planck Society in accordance with the Declaration of Helsinki principles.
5
Turkey Breeder Hen Reproductive Physiology
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The experiment was carried out on a commercial farm of Hybrid XL turkey breeder hens (Grelavi S.A., Poland), localized in the Warmia and Mazury Province in Poland. Samples were collected in 5 stages of birds' life which represented different physiological periods of their reproductive system functioning, that is, in the 32nd week of birds' life (laying onset–sampling 1), 38th wk (laying peak–sampling 2), 44th wk (late laying–sampling 3), 50th and 56th wk, that is, at the end of the laying (sampling 4 and 5, respectively). During each sampling, blood (2 mL/per bird) was collected from the wing vein of turkey breeder hens (n = 5) to test tubes with anticoagulant (BD Vacutainer K2E; BD, Holdrege, NE). Furthermore, after euthanasia of 5 birds and separation of oviducts, mucous membrane samples from the relevant sections (3 parts of magnum [the initial, middle, and final section], isthmus, and 3 parts of uterus [the initial, middle, and final section]) were obtained. Spleen samples (n = 5) were also collected, and eventually all samples were subjected for flow cytometry analysis.
6
PBMC Isolation from EDTA Blood
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Blood was collected into EDTA tubes (BD Vacutainer K2E, Mediq Norge AS, Kløfta, Norway) and kept rotating EDTA blood was loaded onto a SepMate tube (StemCell Technologies, Cambridge, UK) preloaded with Ficoll (GE Healthcare, Oslo, Norway). Plasma was separated according to the manufacturer’s instructions and stored at ‒80°C. PBMC were treated with RBC lysis buffer and stored as pellets at ‒80°C.
7
PBMC and White Blood Cell Isolation
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Blood samples were collected in EDTA tubes (BD Vacutainer K2E). Peripheral blood mononuclear cells (PBMC) and total white blood cells were obtained according to standard protocols. In short, PBMC were obtained using the Ficoll separation technique and whole white blood cells with the simple Pasteur pipette tube technique after spinning samples at 1000 x g as previously described [22 (link)]. Red blood cells were lysed using osmotic lysis buffer (8.3% NH4CL, 1% KHCO3, and 0.04% NA2EDTA in Milli-Q). Upon isolation, PBMC and total white blood cells were resuspended in 0.1% BSA + 2 mM EDTA in PBS and immediately processed for flow cytometry and fluorescence-activated cell sorting (FACS). Isolated PBMC or total white blood cells, not used for direct flow cytometry or FACS, were aliquoted and cryopreserved in complete RPMI (RPMI medium 1640 + glutaMax, Life Technologies) with 10% DMSO (Sigma), 40% Fetal calf Serum (FCS) and stored at − 196 °C until thawing.
8
Placental and Plasma Biomarkers in Preeclampsia
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Ethics approval was obtained from Mercy Health Human Research Ethics Committee (R11/34). Patients presenting to Mercy Hospital for Women (Heidelberg, Victoria) gave written, informed consent for collection of blood during their pregnancy, and placentas following caesarean delivery. Placentas were obtained from patients with established early-onset preeclampsia (<34 weeks’ gestation; n = 43) and gestation matched controls (n = 21). Placentas were processed within 30 min of delivery where tissue was sampled from four quadrants of the placenta and washed in phosphate buffered saline. Placental tissue was processed with RNAlater™ stabilization solution and stored at −80°C for future analysis. Preeclampsia was diagnosed in accordance with the American College of Obstetrics and Gynaecology (ACOG) guidelines (2020) (Obstetricians ACo Gynecologists, 2020 (link)). Refer to Table 1 and Table 2 for patient characteristics.
Whole blood was collected in a 9 ml ethylenediaminetetraacetic acid (EDTA, BD Vacutainer® K2E) tube, centrifuged and plasma obtained from patients delivering at <34 weeks’ gestation with preeclampsia (n = 46), or gestation matched controls (n = 20) who delivered without preeclampsia at term. Plasma was stored at −80°C for further analysis. Refer toTable 3 for patient characteristics.
Whole blood was collected in a 9 ml ethylenediaminetetraacetic acid (EDTA, BD Vacutainer® K2E) tube, centrifuged and plasma obtained from patients delivering at <34 weeks’ gestation with preeclampsia (n = 46), or gestation matched controls (n = 20) who delivered without preeclampsia at term. Plasma was stored at −80°C for further analysis. Refer to
9
Avian Heterophil-Lymphocyte Ratio and Corticosterone Analysis
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Blood was used for the analysis of the heterophil to lymphocyte ratio (H:L ratio ). Blood samples were collected via a heart puncture into a 10-mL EDTA tubes (BD Vacutainer K2E, BD, Franklin Lakes, NJ). The H:L ratio in the blood was analyzed by the method of Kim et al. (2021) (link). The feather samples were also collected from flight feathers to analyze CORT concentrations in the feather. The concentrations of CORT in feathers were analyzed by the method of Lee et al. (2022) .
10
CD14 Expression in Blood Leukocytes
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Venous peripheral blood was collected in ethylenediaminetetraacetic acid (EDTA) treated tubes (BD Vacutainer K2E). To analyse the expression of CD14 on peripheral blood leukocytes, CD14-FITC (Mouse IgG2a κ; BD Biosciences) or Isotype-FITC (Mouse IgG2a; BD Biosciences) were added to 100 μL of whole blood (30 min, room temp.). Then, red cells were lysed (BD FACSTM Lysing Solution). Finally, 10 000 events were collected and analysed by means of a BD FACSCalibur flow cytometer. Data were examined using WinMDI 2.9 software (Joseph Trotter, La Jolla, CA. USA).
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