Peripheral blood from three healthy 21–28 years old females was used for the experiments. Informed consent was obtained from each human subject for the use of blood cells in this study. The blood was withdrawn using Vacuette® K2EDTA blood collection tubes (Greiner Bio-One, Frickenhausen, Germany), followed by centrifugation in a ficoll-verografin density gradient (Histopaque by Sigma-Aldrich, Poole, UK) according to the manufacturer’s recommendations. Lymphocytes were then rinsed and resuspended in phosphate buffered saline (PBS) at a concentration of 106 cells/mL. Two hundred μL of cell suspension was mixed with 600 μL of 1% low melting point agarose (Thermo Scientific, Rockford, IL, USA) in PBS at pH 7.4 and 37.5 °C. Then 75 μL of the lymphocyte-agarose mix was dispensed onto microscope slides pre-coated with 1% normal melting point agarose (Thermo Scientific, Rockford, IL, USA). Following mounting with coverslips, the slides were chilled at 4 °C for 10 min to allow the solidification of the lymphocyte-agarose layer. Slides were then removed and agarose-embedded cells were exposed to X-rays.
Vacuette k2edta blood collection tubes
Manufactured by Greiner
Sourced in Germany
Vacuette® K2EDTA blood collection tubes are designed for the collection and transportation of venous blood samples. They contain K2EDTA as an anticoagulant additive.
Automatically generated - may contain errors
2 protocols using vacuette k2edta blood collection tubes
1
Lymphocyte Comet Assay Protocol
2
DNA Methylation Analysis Using Infinium MethylationEPIC BeadChip
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Whole blood for DNA extraction was collected in VACUETTE K2EDTA blood collection tubes (Greiner Bio‐One International). DNA methylation was analyzed using the Infinium MethylationEPIC BeadChip, which quantitatively interrogates DNA methylation at >850 000 positions (CpGs) genome‐wide with single nucleotide resolution. The EPIC BeadChips were processed at the LIFE & BRAIN laboratory according to the manufacturer's instructions. All DNA methylation analyses were carried out using the R programming language (http://www.r‐project.org/ ). Preprocessing and quality assessment were performed using functions implemented in the minfi package.21 First, normalization was performed with precrocessQuantile. Then, the data were preprocessed and filtered to remove probes with unreliable measurements (detection p‐values > .01, n = 20 283), probes located on the sex chromosomes (n = 18 654), probes with overlapping single nucleotide polymorphisms (n = 27 348), cross‐reactive probes (n = 39 112), and non‐CpG probes (n = 2458),22 , 23 resulting in a final dataset consisting of 760 462 probes and 172 samples.
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