Microglia-Neuron Interactions in Hemoglobin-Induced Toxicity

Primary microglia were prepared from postnatal day 1 C57BL/6J mice as previously reported [13 (link)]. Primary neurons were prepared from a day 15 embryonic cortex obtained from the pregnant C57BL/6J mice as previously reported [17 (link)]. All experiments were carried out 24 h after cells were seeded. The cells were treated with hemoglobin (Hb) (Sigma, St. Louis, MO, USA) diluted in culture medium or combined with SP600125 (Abcam, Cambridge, USA) diluted in DMSO for 24 h. The vehicle was used as the control. The supernatants were removed and replaced with fresh DMEM for another 24 h. Then, microglia were collected for assays. The conditioned medium of microglia was collected and added to neurons which were cultured in DMEM/F12 supplemented with 10% FBS for 24 h. Then, the cells were harvested for experiments.