Vacutainer spray coated edta tubes

Human blood samples were purchased from the Bonfils Blood Center Headquarters of Denver Colorado. Our use of these deidentified samples was determined to be “Not Human Subjects” by our Institutional Review Board. Biopsies were collected as unfractionated whole blood from apparently healthy donors, though samples were not tested for infection. Samples were approximately 10 mL in volume, and collected in BD Vacutainer spray-coated EDTA tubes. Following collection, samples were stored at 4° until processing, which occurred within 5 hr of donation. To remove plasma from the blood, samples were put in 50 mL conical tubes (Corning #430828) and centrifuged for 10 min at 515 rcf. Following centrifugation, plasma was aspirated and 200 mL of 4° hemolytic buffer (8.3g NH₄Cl, 1.0g NaHCO₃, 0.04 Na₂ in 1L ddH₂O) was added to the samples and incubated at 4° for 10 min. Hemolyzed cells were centrifuged at 515 rcf for 10 min, supernatant was aspirated, and pellet was washed with 200 mL of 4° PBS. Washed cells were centrifuged for at 515rcf for 10 min, from which gDNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen REF 69504).

Plasma was obtained from healthy donors after they signed a voluntary and informed study participation form. Blood was collected in BD Vacutainer spray-coated EDTA tubes; plasma was immediately separated from the blood, aliquoted, and stored at −80 °C. Before use, plasma was slowly thawed at 4 °C. In order to remove cells and cellular detritus, plasma was centrifuged at 400× g—10 min, 800× g—10 min, 1500× g—10 min, and 17,000× g—30 min. Each time, the supernatant was carefully replaced in a new tube. After the last centrifugation, the supernatant was filtered through a 0.2 mkm PES syringe filter to obtain pellet pure plasma (PPP). Plasma treated in this way was used in all experiments described in this study.