Oxidative Stress Induction in CD34+ Cells

CD34+ cells were seeded in 24-well plates at 5 × 10⁵ cells/mL in Iscove’s modified Dulbecco’s medium (IMDM) (EuroClone, Pero, Milan, Italy) containing 10% Human Serum (Sigma-Aldrich, St. Louis, MO, USA), SCF (50 ng/mL), FLT3-ligand (50 ng/mL), TPO (20 ng/mL), IL-6 (10 ng/mL) and IL-3 (10 ng/mL) (all from Miltenyi Biotec, Bergish Gladbach, Germany), as previously described [29 (link)]. To induce oxidative stress, CD34+ cells were treated with Melittin, the main constituent and principal toxin of bee venom with powerful oxidant activity [30 (link)]. Briefly, CD34+ cells were seeded at 1 × 10⁶ cells/mL in IMDM medium supplemented with 10% Human Serum and Melittin 5 µg/mL (Sigma-Aldrich, St. Louis, MO, USA) for 6 and 24 h at 37 °C in a humidified atmosphere with 5% CO₂.