Lgm 3 culture medium

Peripheral blood samples were collected from healthy donors in 9 ml EDTA vacutainer tubes. Blood collection was approved by the local SCK CEN Ethics Committee and was carried out in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2000. Prior to blood donation all the donors involved in the present study signed an informed consent form. Within 30-60 min of blood drawing, PBMCs were isolated by centrifugation on Histopaque-1077 density gradient (Sigma-Aldrich, Bornem, Belgium) according to the manufacturer’s instructions. Isolated cells were suspended at a density of 10⁶ cells/ml in LGM-3 culture medium (Lonza, Walkersville, MD, USA) and were allowed to equilibrate to culture conditions at 37°C in a humidified 5% CO₂ atmosphere.

Venous blood from three healthy donors was taken at the Centre for Radiation, Chemical and Environmental Hazards, UKHSA (Chilton, UK) with informed consent and the ethical approval of the West Midlands–Solihull Research Ethics Committee (REC 14/WM/1182) for these experiments.
From whole blood samples (20 mL per donor), human peripheral blood mononuclear cells (PBMCs) were isolated using a density gradient centrifugation of peripheral whole blood using Lymphoprep (STEMCELL Technologies, Cambridge, UK). Briefly, blood was diluted with 1:1 volume of Dulbecco’s phosphate-buffered saline (DPBS) and layered on top of the Lymphoprep solution (15 mL of Lymphoprep for 10 mL of diluted blood). After a centrifugation at 800× g at 22 °C for 20 min, the cloudy PBMC layer between a top layer of plasma and a bottom layer of polymorphonuclear cells was collected and transferred to a new tube to proceed with DPBS washes (300× g at room temperature for 10 min). After the isolation, 1.5–2 × 106 PBMCs were seeded in T25 flasks and maintained in LGM-3 culture medium (Lonza, Slough, UK) for 0–72 h at 37 °C in a humidified 5% CO2 atmosphere.

Blood from nine healthy donors was collected and exposed to 0 Gy or 2 Gy (10 mL of blood each, dose rate 0.5 Gy/min). After irradiation, the peripheral blood mononuclear cells (PBMCs) were isolated using Histopaque-1077 (Sigma Aldrich, Poole, Dorset, UK) and maintained in LGM-3 culture medium (Lonza, Slough, UK) at 2 × 10⁶ cells ml/mL for 24 h at 37 °C in a humidified 5% CO₂ atmosphere. After 24 h, the PBMCs were pooled in groups of three donors each, and the RNA was extracted using the RNeasy Midi kit (Qiagen, Manchester, UK). The quantity of isolated RNA was determined by spectrophotometry with a ND-1000 NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) and quality was assessed using a Tapestation 220 (Agilent Technologies, CA, USA). Venous blood was taken at the Centre for Radiation, Chemical and Environmental Hazards Public Health England (Chilton, UK) with informed consent and the ethical approval of the West Midlands Solihull Research Ethics Committee (REC 14/WM/1182).