4.8. NLT Cell Culture and Treatment Protocols

Mouse GnRH-expressing neuronal cells (NLT) was maintained by our laboratory. NLT cells were grown in DMEM (Sigma Aldrich, St Louis, MO, USA) contain 10% FBS (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C under a humidified atmosphere of 95% air and 5% CO₂. To determine the neurotoxicity of hemin, various concentrations (0, 10, 30, 60, 90 and 120 µM) of hemin (Sigma-Aldrich) were added to NLT cells and treated for 6 h. Of these concentrations, 60 µM (LD50) was selected and used in the subsequent experiments. Control groups were treated with vehicle (DMSO) only. Cell viability and Live/Dead staining were performed 6 h after treatment. Three wells were used for each group. Experiments were repeated at least 3 times.