Blood samples were collected in sodium-heparin tubes (Greiner Bio-One, Germany) followed by isolation of peripheral blood mononuclear cells (PBMCs) as described before [19 (link)]. PBMCs were thawed, centrifuged for 5 min (1500 rpm, 4 °C) and resuspended in 100 μl sort‐buffer (PBS pH 7.4, 2 mM EDTA 1 M, 0.5% BSA). For membrane staining, cells were incubated for 15 min in the dark with anti‐CD14 (APC/Cy7; Becton Dickinson Biosciences, San Diego, USA), anti-BDCA‐4 (PE; Miltenyi Biotec, Bergisch Gladbach, Germany), anti‐CD123 (PE-Cy7; eBioscience, San Diego, USA), anti‐CD3 (PerCP-Cy5; Becton Dickinson Biosciences), and anti‐CD19 (APC; Becton Dickinson Biosciences). Cells were sorted using a FACSAria III cell sorter (BD Bioscience) and analyzed using FlowJo Sofware (TreeStar Inc., Ashland, USA).
Sodium heparin tubes
Manufactured by Greiner
Sourced in Germany
Sodium-heparin tubes are laboratory equipment used for the collection and storage of blood samples. They contain sodium heparin as an anticoagulant, which prevents the blood from clotting during the collection and processing stages.
Automatically generated - may contain errors
Lab products found in correlation
Gentamycin, by Duchefa Biochemie (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
Anti cd19 apc, by BD (1 mentions)
Anti bdca4 pe, by Miltenyi Biotec (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Phagoburst, by BD (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Foetal bovine serum (fbs), by Biowest (1 mentions)
Hyclone, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Nh4cl, by Merck Group (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
4 protocols using sodium heparin tubes
1
Isolation and Characterization of PBMCs
2
Isolation of Neutrophils from Healthy and CGD Donors
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Peripheral blood from HDs and from two CGD patients (twin boys) was collected in sodium-heparin tubes (Greiner Bio-One). All blood donors gave informed consent. Patients with CGD were recruited in the outpatient clinic of the department of Paediatric Immunology and Infectious Diseases of the UMC Utrecht. Diagnosis of CGD was determined functionally by absence of respiratory burst upon stimulation in neutrophil (Phagoburst, BD Bioscience) and genetically by Sanger sequencing of the NCF1 gene showing a pathogenic homozygous mutation (delta GT). Neutrophils were isolated by Ficoll-Paque (GE Healthcare) density gradient centrifugation, after which erythrocytes were lysed in ammonium chloride buffer (155 mM NH4Cl; 10 mM KHCO3; 0.1 mM EDTA in double-distilled H2O; pH = 7.2). Cells were resuspended in RPMI 1640 (Life Technologies) supplemented with 10% (v/v) heat-inactivated (HI) foetal bovine serum (FBS) (Biowest) and 50 U/ml Penicillin-Streptomycin (referred to as RPMI 10% hereafter). Purity of isolated neutrophils was analysed using the CELL-DYN Emerald and was >88%. All experiments were performed in accordance with relevant guidelines and regulations approved by the Medical Ethical Committee of the University Medical Centre Utrecht.
3
Isolation of Neutrophils from Healthy Donors
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Blood was collected from healthy volunteer donors after obtaining informed consent into sodium heparin tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands). Neutrophils were isolated using density gradient followed by erythrocyte lysis, as previously described [23 (link)]. Neutrophils were then resuspended in IMDM (Gibco; Thermo Fischer Scientific Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS; Hyclone; Thermo Fischer Scientific Inc., Waltham, MA, USA) and gentamycin (86 µg/mL; Duchefa Biochemie B.V., Haarlem, The Netherlands) and used immediately. Neutrophil purity was analyzed by flow cytometry and was always >97%.
4
Isolation and Purification of Human Neutrophils
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Blood was collected from healthy volunteer donors after informed consent into sodium heparin tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands). Blood was diluted with Hanks balanced salt solution (HBSS, Sigma-Aldrich Inc., St. Louis, MO, USA) and granulocytes and erythrocytes were separated from peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation on Lymphoprep (d = 1.077 ± 0.001 g/mL; Axis-Shield, Oslo, Norway). Erythrocytes were lysed in ice-cold erythrocyte lysis buffer (containing 0.155 M NH4Cl (Sigma-Aldrich, Inc., St. Louis, MO, USA), 1 mM KHCO3 and 80 µM EDTA (both Merck KGaA, Darmstadt, Germany), dissolved in sterile water, pH 7.3) for 10 min on ice. Subsequently, neutrophils were cleared from remaining erythrocytes with a second lysis step for 5 min on ice, and washed twice in PBS. Neutrophils were then resuspended in IMDM (Gibco; Thermo Fischer Scientific Inc, Waltham, MA, USA) supplemented with 10% heat inactivated (HI) fetal bovine serum (FBS; Hyclone; Thermo Fischer Scientific Inc, Waltham, MA, USA) and gentamycin (86 µg/mL; Duchefa Biochemie B.V., Haarlem, The Netherlands) and used immediately. Neutrophil purity was analyzed by flow cytometry and was always >97%.
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