Isolation of Normal Mononuclear Cells

Normal mononuclear cells were offered by Dr. Marwan El-Sabban’s Lab at the American University of Beirut (AUB) as a kind gift. The normal MNCs were obtained originally from bone marrow aspirate leftovers of healthy patients attending AUB Medical center (AUB-MC). BM aspirates were centrifuged on Ficoll/Hypaque (GE Healthcare Life Sciences, Uppsala, Sweden), a density gradient step to separate MNCs from red blood cells and neutrophils. Then the buffy coat was aspirated and seeded in petri dishes using Dulbecco’s modified Eagle’s medium (DMEM)-low glucose (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% FBS (Gibco, Dublin, Ireland) and 100 U/mL penicillin and 100 µg/mL streptomycin (Lonza, Basel, Switzerland) in a humidified incubator at 37 °C and 5% CO₂. One week later, the cells in suspension were collected as a purified MNC population and cultured in the same conditions as detailed by Zibara et al. [25 (link)].