2.2. Primary Neural Cultures

Primary astrocytes were isolated from newborn (1–2 days old) Fmr1 WT or KO mice using the differential adhesion method [14,23]. In our preparations, over 95% of the adherent cells were astrocytes as demonstrated by anti-glial fibrillary acidic protein (GFAP) immunostaining. To obtain astrocyte-conditioned medium (ACM), astrocytes were first cultured in 24-well culture plates for 24 h in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum. Cultures were washed extensively with Hank’s balanced salt solution (HBSS; Invitrogen, Carlsbad, CA, USA) and changed to neurobasal medium with B27 supplementation (NB/B27; Invitrogen, Carlsbad, CA, USA) by adding 500 μM glutamine. The NB/B27-based medium was conditioned for 7 days and collected after brief centrifugation. During this period, the medium had few effects on astrocyte proliferation and cell viability [24]. Prior to exposure on neuron culture, the ACM had an osmolarity of 292 mOsm/kg and pH of 6.80.

The cortical neurons were prepared from 15-day-old embryos (E15) of WT mice according to a previously described method [14,25]. Neurons were cultured in NB/B27 with half of the medium changed every 3 days. For ACM culturing, neurons were plated for 4 h and then replaced by a 1:1 mixture of ACM and neuronal maintenance medium.