Mice were anesthetized and blood samples were obtained by cardiac puncture and were collected either in a K3 EDTA vacutainer (cat# 367,861, BD) or in an Eppendorf tube. Samples were centrifuged at 2000 RPM for 20 min and blood plasma was collected as supernatant, aliquoted and stored at—80 °C for downstream analyses. Standard procedures were followed according to the manufacturer’s instructions and blood cytokines levels were analyzed by standard ELISA kits for IFNγ (cat#: MIF00, R&D), IL-6 (cat #: M6000B) and IL-10 (cat#: BMS614-2). In addition, MCP-1 (Cat# AF-479, R&D) levels were analyzed by Western blotting.
Vacutainer k3 edta
Manufactured by BD
Sourced in United Kingdom, United States
The BD Vacutainer K3 EDTA is a blood collection tube that contains the anticoagulant K3 EDTA. It is designed to prevent blood clotting, allowing for accurate analysis of blood samples.
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7 protocols using vacutainer k3 edta
1
Blood Cytokine Profiling in Mice
2
Circulating Cell-free DNA Isolation
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Blood samples were collected in a K3 EDTA Vacutainer (BD, Plymouth, UK) tubes and stored at 4–8°C for up to 24 h before plasma separation. The plasma was separated by two step centrifugation at 2200 g and 16000 g each for 10 min and stored at –20°C until further processing. The cell-free DNA was isolated from 1.8 ml of maternal plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). A final elution volume of 85 μl and 56 μl was used for samples analyzed during the validation phase on Ion Torrent PGM and MiSeq, respectively. For physical size selection protocol the DNA was isolated from second plasma aliquot from 10 samples with chromosome 21 trisomy and eluted to 56 μl of molecular biology grade water.
3
Blood Sampling and Plasma Extraction
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For longitudinal time points, blood was withdrawn from saphenous veins, whereas cardiac blood samples were taken at the endpoint. For mouse saphenous blood sampling, 40 μl was pipetted into 200 μl EDTA (4.5 mM, pH 8.0) in phosphate-buffered saline (PBS). For cardiac blood sampling, blood (typically, 200 µl) was collected from terminally anaesthetised mice into K3-EDTA vacutainers (BD Biosciences). Blood was centrifuged at 1000 g for 10 min, and the plasma supernatant was centrifuged at 1000 g for a further 10 min. Blood samples were processed promptly to reduce haemolysis; samples with evidence of haemolysis by visual inspection were excluded from further analysis. Plasma volumes were adjusted to 200 µl with PBS before extraction of cfDNA.
4
Femoral Blood Collection and Plasma Isolation
5
Blood Sample Collection and Processing for CRC Research
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Blood samples were collected in Sarstedt S-Monovette K3 EDTA or BD Vacutainer K3 EDTA tube prior to bowel preparation for bowel colonoscopy (BLITZ study), or prior to large bowel surgery or neoadjuvant therapy (239 CRC cases from the clinical setting). The samples were transported to the laboratory while preserving a cold chain and were centrifuged at 2000–2500× g for 10 min, aliquoted and stored at −80°C until further use. Details on the SOPs have also been described previously.19 (link)
6
Blood Fractionation from Median Cubital Vein
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Peripheral blood was obtained from the median cubital vein using BD vacutainer K3 EDTA (BD, USA). Samples were stored at +4 °C and subjected to fractionation no later than 2 h after collection.
7
Canine Immune Subpopulation Analysis
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Blood samples were collected in a BD vacutainer K3EDTA. Samples were incubated with human FcR Blocking (Miltenyi; Cologne, Germany) for 10 min at room temperature and incubated for 20 min at 4°C with directly conjugated antibodies diluted in phosphate-buffered saline with 1% fetal bovine serum. Samples were incubated with Quicklisis buffer (Cytognos; Salamanca, Spain) to lyse erythrocytes. The acquisition and analysis were conducted in a MacsQuant10 Flow Cytometer (Miltenyi). The antibodies employed and the flow cytometry gating strategy of the immune subpopulation analysis in dog-derived samples have been previously described.6 (link)
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