L glutamine g3126

After three days in culture, the medium of neuron-astrocyte mixed cultures was changed to non-essential amino acid medium, consisting of MEM (M0268, Sigma-Aldrich, St. Louis, MO, USA) with 2.2 g/L NaHCO₃ (S/4240/60, Fisher Scientific, Loughborough, UK), pH 7.3, and supplemented with 1 g/L D-glucose anhydrous (G/0450/60, Fisher Scientific, Loughborough, UK), 0.292 g/L L-glutamine (G3126, Sigma-Aldrich, St. Louis, MO, USA), 0.11 g/L pyruvic acid (P2256, Sigma-Aldrich, St. Louis, MO, USA), 1% MEM non-essential amino acid solution (M7145, Sigma-Aldrich, St. Louis, MO, USA) and 10% heat-inactivated fetal bovine serum (S0615, Biochrom, Berlin, Germany). The choice of this medium was based on its compatibility with neurons, astrocytes, and microglial cells. Twelve hours later the cells were exposed to 1-methyl-4-phenylpyridinium neurotoxin (MPP⁺, 10 µM; D048, Sigma-Aldrich, St. Louis, MO, USA). After thirty-six hours, the conditioned media (CM) were collected from both control neuron-astrocyte cultures (NA-CM) and neuron-astrocyte cultures exposed to MPP⁺ (NA(MPP⁺)-CM) and stored at −80 °C until use.

Retinal cell line 28 (R28) is an adherent retinal precursor cell line derived from neonatal Sprague-Dawley rat retina, which was immortalized by the 12S E1A gene and commonly used to study the function and neuroprotection of RGCs in vitro (19 (link)). In this study, R28 cells were offered by the Department of Anatomy and Neurobiology (Central South University, Changsha, China). Cells were cultured in low glucose DMEM (11885084; Gibco, Carlsbad, USA) with 10% fetal calf serum (0500; ScienCell, San Diego, USA), 1% L-glutamine (G3126; Sigma, St. Louis, USA), 1% non-essential amino acids (M7145; Sigma, St. Louis, USA), and 1% penicillin-streptomycin (60162ES76; Yeasen, Shanghai, China). To establish the Oxygen-Glucose Deprivation (OGD) model, an in vitro model of retinal IR, R28 cells were incubated in a glucose-free DMEM (11966025; Gibco, Carlsbad, USA) with an anoxic environment (95% N₂, 5% CO₂) for 2 h, then the cells were cultured in normal medium and reoxygenated (21% O₂, 5% CO₂) for 2 h before being examined (Figure 1B).