Fresh whole blood was collected in ACD solution A vacutainer (Becton Dickinson, Franklin Lakes, NJ) tubes for each participant. CD14+ monocytes were immediately separated within 120 minutes of collection using whole blood CD14 microbeads and the AutoMACSPro (Miltenyi Biotec, San Diego, CA). Total RNA was extracted immediately following cell separation using the mirVana miRNA isolation kit (Life technologies, Carlsbad, CA). RNA quantity was assessed on the Qubit Fluorometric quantitation system (Life Technologies). RNA quality was also assessed on a Bioanalyzer (Agilent, Wilmington,DE) prior to running the TaqMan ®Array Human MicroRNA Card A (Life technologies).
Vacutainer acd solution a
Manufactured by BD
Vacutainer ACD Solution A is a laboratory blood collection tube that contains an anticoagulant, the Acid-Citrate-Dextrose (ACD) solution. The ACD solution prevents the blood from clotting, allowing for proper sample analysis.
Automatically generated - may contain errors
Lab products found in correlation
Vacutainer venous blood collection tubes sst, by BD (2 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
Qubit fluorometric quantitation system, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Lymphoprep density gradient centrifugation, by STEMCELL (1 mentions)
Hetasep, by STEMCELL (1 mentions)
Easysep neutrophil enrichment kit, by STEMCELL (1 mentions)
K2edta vacutainer, by BD (1 mentions)
7 protocols using vacutainer acd solution a
1
Monocyte Isolation and microRNA Profiling
2
Isolation of Human Neutrophils
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Neutrophils were isolated from fresh whole blood collected in ACD solution A vacutainer (Becton Dickinson, Franklin Lakes, NJ) tubes. Briefly, neutrophils were isolated through density gradient centrifugation as described (30 (link)). Cells were then further purified using magnetic-bead based negative selection with the MACSexpress Human Whole Blood Neutrophil Isolation kit (Miltenyi Biotec, Germany), followed by hypotonic lysis of remaining red blood cells. Neutrophil purity was assessed by flow cytometry (see below) and was typically >98%.
3
Isolation of PBMCs from Blood
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Blood samples were collected from healthy female donors or female breast cancer patients in an ACD Solution A Vacutainer (BD; Cat. No. 364606). PBMCs were collected using a density gradient medium, Ficoll-Paque Plus (GE Healthcare Life Sciences), or Lymphoprep (StemCell). The buffy coat, containing lymphocytes, was collected and cells were counted. PBMCs were either used for immediate NK cell expansion or cryopreserved [30 ].
4
Isolation and Stimulation of Human PBMCs
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Peripheral blood was collected with written-informed consent and with approval by the Hamilton Integrated Research Ethics Board at McMaster University. Peripheral blood was collected in an ACD solution A vacutainer (BD Biosciences) to prevent clotting. Blood was then diluted with 2% FBS in PBS and then separated using a Lymphoprep density gradient centrifugation method (Stem Cell Technologies). Isolated PBMCs were then used for in vitro assays or funneled through a CD56+ NK cell isolation kit purchased from StemCell Technologies. Cells were plated at 2 × 105 cells/mL and then stimulated with 100 U of IFN-β for 12 or 18 h (as specified in the figure legends) and subsequently stimulated with either 250 ng/mL IL-15 for 24 h or examined for Axl, Mer, or Tyro3 expression via flow cytometry. Supernatants were collected from cells that were further stimulated with IL-15 and examined for IFN-γ production as discussed below.
5
Isolation of Human Neutrophils
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To isolate human neutrophils, peripheral blood was drawn into tubes containing trisodium citrate, citric acid, and dextrose (Vacutainer ACD Solution A, BD). Autologous serum, used for culture of cells was obtained by drawing blood into BD Vacutainer™ Venous Blood Collection Tubes SST, followed by centrifugation at 2500 × g for 30 min and serum collection. All blood donors provided written informed consent. For normal human blood samples, 10mls human blood was supplemented with GM-CSF (10 ng/ml) for 30 min at 37 °C followed by addition of 30 μg FITC-IgG isotype control or FcγRIIIB (3G8)-FITC-Ova conjugate for 2 h at the indicated concentrations. Blood was then incubated with Hetasep (STEMCELL Technologies) according to manufacturer protocols to deplete red blood cells and enrich leukocytes. Neutrophils were isolated from the leukocyte-rich plasma layer using a Easysep Neutrophil enrichment kit (STEMCELL Technologies). Neutrophil purity was evaluated using CD15, CD11b, CD66b, and lineage markers (CD3, CD19, and CD56) Supplementary Tables 4 and 6 .
6
Healthy Donor Blood Collection Comparison
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The study was approved by the ethical committee of Ghent University Hospital (EC/2015/0260) and performed according to the guidelines and regulations of the Declaration of Helsinki. Venous blood was collected from 10 healthy overnight fasting donors, which did not use any medication or suffer from any acute or chronic disease at the time of venipuncture. A 21‐gauge needle was used for venipuncture after applying a light tourniquet. The first few milliliters of blood were discarded. Blood from the 10 healthy donors was collected in four collection tubes containing different anticoagulants and/or preservatives: citrate (Vacuette sodium citrate 3.2%, volume 9 ml, Greiner Bio‐One B.V.), anticoagulant citrate dextrose solution A (ACD‐A; Vacutainer ACD Solution A, volume 8.5 ml, BD), ethylenediaminetetraacetic acid (EDTA; Vacutainer K2EDTA, volume 10 ml, BD), and EDTA separator (Vacuette K2EDTA with separator, volume 8 ml, Greiner Bio‐One B.V.). The drawing sequence of collection tubes was randomized.
7
Blood Collection and Serum Extraction
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Peripheral blood was drawn into tubes containing trisodium citrate, citric acid and dextrose (Vacutainer ACD Solution A, BD). Serum was obtained by drawing blood into BD Vacutainer Venous Blood Collection Tubes SST, followed by centrifugation at 2500xg for 30 min and removal of the resulting supernatant.
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