Neurobasal a b27

Newborn C57BL/6 mice (within 24 h) were procured from the animal facility of the University of Macau. The whole body was disinfected with 75% alcohol and the brain was surgically removed and stored into cold HBSS (calcium- and magnesium-free) balance solution. The whole hippocampal region was dissected using a glass rod that was bent on both sides. The hippocampus was cleared of the blood and the mixed blood vessels by washing three times with HBSS. Then, the hippocampus was chopped into 1 mm³ pieces using scissors, and after washing three times with HBSS the tissue was digested with 0.125% trypsin at 37 °C for 15 min. The enzymatic digestion was stopped with 10% FBS and 5 mL of neurobasal A (Gibco, Carlsbad, CA, USA) was added to the digested hippocampal tissue in a 15 mL centrifuge tube. The turbid tissue supernatant was collected in another 15 mL centrifuge tube and centrifuged at 1000 rpm for 10 min. The resulting cell pellet was resuspended in neurobasal A/B27 (Gibco, Carlsbad, CA, USA), seeded in poly-D-lysine -treated plates at a density of approximately 1 × 10⁵ cells/mL and incubated for growth at 37 °C in a 5% CO₂ humidified atmosphere.

Newborn C57BL/6 mice were procured from the animal facility of University of Macau. The whole body was disinfected with 75% alcohol and the brain was surgically removed and stored into cold HBSS (Ca²⁺, Mg²⁺ free) balance solution. The whole hippocampus region was dissected using a glass rod which was bent on both sides. The hippocampus was cleared of the blood and the mixed blood vessels by washing thrice with HBSS. Then the hippocampus was chopped into 1 mm³ pieces using scissors and after washing thrice with HBSS the tissue was digested with 0.125% of trypsin at 37 °C for 15 min. The enzymatic digestion was stopped with 10% FBS and 5 mL of Neurobasal A (Gibco, USA) was added to the digested hippocampus tissue in a 15 mL centrifuge tube. The turbid tissue supernatant was collected in another 15 mL centrifuge tube and centrifuged at 1000 rpm for 10 min. The resulting cell pellet was resuspended in Neurobasal A/B27 (Gibco, Carlsbad, CA, USA) and seeded in poly-D-lysine treated plates at a density of about 1–2 × 10⁵ cells / mL and incubated for growth at 37 °C in 5% CO₂ humidified atmosphere.

Slices of 350 μm thickness were made using McIlwain tissue chopper (Mickle Laboratory Eng. Co., Surrey, United Kingdom), from the hippocampi of Sprague-Dawley rats of post-natal days 7–8. Tissue cultures were then transferred to a poly-D-lysine (PDL) coated substrates. For substrate microwire recordings (s-MW), 6-well plates (Falcon) were used. MEA recordings were collected from micro-electrode arrays (MEA) (60MEA 200/30 IR-TI, Multichannel systems). For optical recordings, 35mm petri-dishes (Falcon) were used as substrates. In the case of single inserted electrode recordings (i-MW), slices were placed on glass coverslips pre-coated with PDL and cultured in 6-well plates. Slices were maintained in a humidified 37 °C incubator with 5% CO₂ on a rocking platform. A serum free culture medium consisted of Neurobasal-A/B27, 30 μg/ml gentamicin, and 0.5 mM GlutaMAX (Invitrogen). Culture media was changed twice per week. All animal use protocols were approved by the Institution Animal Care and Use Committee (IACUC) at Lehigh University and were conducted in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.