Alanyl glutamine

Primary neuronal cultures were obtained from hippocampi dissected from C57BL/6S E 18 mice (Harlan, Udine, Italy). Animals were maintained in a pathogen free animal facility. All experiments were performed in strict accordance with experimental procedures approved by the Italian Ministry of Health.
Embryos were removed and dissected under sterile conditions. Hippocampi were dissociated by enzymatic digestion in trypsin (0.125% for 30 min at 37 °C). Trypsin activity was blocked by adding complete media (NeurobasalTM (Gibco) supplemented by B27 (2%, Gibco), alanyl- glutamine (2 mM, Gibco), penicillin/streptomycin (both 1 mM, Sigma) containing 10% fetal bovine serum (FBS, Gibco). After trypsinization, tissues were rinsed in complete media without FBS, and dissociated with a plastic pipette. Neurons were plated either on glass-bottom Petri dishes for long-term imaging (density of 20,000 neurons on 10 mm diameter glass bottom); or on glass coverslips for immunocytochemistry (density of 30,000 neurons on 18 mm diameter coverslips); or on glass coverslips (square #3D) for chemotaxy assay (density of 50,000 neurons on 20 × 26 mm coverslip); or on plastic Petri multi wells dishes for immunoblot (density of 350,000 neurons on 30 mm diameter dish). After 2 h from plating, 2 ml serum-free glial conditioned medium was added.

In brief, primary culture was obtained from mouse hippocampus on the 18th embryonic day, dissected under sterile condition, and detached with 0.125% trypsin at 37 °C for 20 min. Then, complete medium (Neurobasal2, Gibco, Grand Island, NY, USA) containing 2% B27 (Gibco), 2 mM alanyl-glutamine (Gibco), 10% fetal bovine serum (FBS), and 1 mM penicillin/streptomycin (Sigma, St. Louis, MO, USA) were applied to terminate the detachment. Tissues were washed by FBS-free complete medium and dissociated using a plastic pipette. Neurons were spread on a petri dish with a glass bottom at a density of 0.25–1 × 10⁵ cells/mL and allowed to stand for 2 h in the incubator for attachment. Then, a serum-free medium was added until the volume reached 2/3. Primary hippocampal neurons HT22 (CL-0595; Procell Life Science & Technology Co., Ltd., Hubei, China; https://www.procell.com.cn/view/4535.html) were used for in vitro experiments in this study. HT22 cells were cultured in the Dulbecco’s modified Eagle’s medium containing 10% FBS, 2 mM L-alanyl-L-glutamate, 0.24% hydroxyethyl piperazine ethane-sulfonic acid (HEPES), 0.375% sodium bicarbonate, 100 U/mL penicillin, and 100 mg/mL streptomycin.