Dmem low glucose dmem lg

Mesenchymal stem cells were isolated from the adipose tissues in the subcutaneous inguinal region of 8–10 weeks old male Sprague–Dawley (SD) rats (about 250 g), following protocol previously reported 22 (link). In general, the adipose tissues were digested with 0.075% type I collagenase (Sigma-Aldrich, St Louis, MO, USA) in a 5% CO₂ incubator at 37°C for 1 hr. The resulting mixture was filtered through a 140 μm nylon mesh, followed by centrifuged at 200 × g for 10 min. at 4°C. After washing, the pellet was loaded onto a Percoll density gradient (1.077 g/ml) and finally centrifuged at 100 × g for 20 min. at 4°C. The resulting interphase was washed and cultured in DMEM-low glucose (DMEM-LG; Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (MDgenics, St. Louis, MO, USA) in a 5% CO₂ incubator at 37°C. After four to five passages expansion, the MSCs were characterized by flow cytometry before transplantation.

Neurobasal medium, α-MEM, RPMI medium, DMEM low glucose (DMEM-lg), DMEM/F-12, fetal bovine serum (FBS), L-glutamine, dispase II, Glutamax, antibiotics, trypsin/EDTA, human neurotrophic factors, N2, and B27 were all purchased from Gibco™ Thermo Fisher Scientific Inc. (MA, USA). Fibronectin, heparin, gelatin, nonessential amino acids, Hoechst 33258, and normal goat serum (NGT) were acquired from Sigma-Aldrich, Merck (St. Louis, MO, USA). The following primary antibodies were used: mouse anti-TUJ1 (SC-80016) and rabbit anti-MAP2 (SC-20172) obtained from Santa Cruz Biotechnology (CA, USA); rabbit anti-GABAB (SC-376282), ENO2 (GTX113428), TAU (GTX116044), and mouse anti-NF-H (GTX27795) from GeneTex Inc. (CA, USA); and rabbit anti-NeuN (MAB377) from Merck-Millipore (Darmstadt, Germany). Secondary antibodies against goat anti-mouse IgG conjugated to Alexa Fluor 488 and goat anti-rabbit IgG conjugated to Alexa Fluor 568 were obtained from Molecular Probes, Thermo Fisher Scientific Inc. (MA, USA). Small molecules I-BET151, CHIR99021, forskolin, RepSox, Y-27632, and dibutyryl cAMP were purchased from Sigma-Aldrich, Merck (St. Louis, MO, USA).