Sema 3A Effects on Fetal Brain Microglia

Human fetal brain-derived primary cultures of microglia (HMC3, 37,089–01), purchased from Celprogen Inc. (Benelux, NL), were cultured in Essential medium 8 (ThermoFisher, Milan, IT), that is routinely used for stem cells grow and expansion and we previously used to keep in culture NP [37 (link), 39 ]. DNA transfection was performed by incubating 10 µg/ml of Sema 3A with 15 µl of Lipofectamine 2000 (Invitrogen, ThermoFisher, Milan, IT), according to the manufacturer protocols. After 20 min, fresh media was added, and cells were left in culture for 48 h. 10 µg/ml of GFP-empty vector was used as transfection positive control.
Media from Sema 3A, GFP or non-transfected microglia was collected 48 h after transfection and transferred to NP culture. In order to minimize events related to changes in growth conditions, NP were cultured in Essential medium 8 (ThermoFisher, Milan, IT) at least 48 h before the experiment.