Neurobasal a b27 medium

All animal-use protocols were in accordance with the guidelines of the National Institutes of Health and our Institution for Comparative Medicine on the use of laboratory animals, and approved by the Subcommittee on Research and Animal Care. Hippocampal slices were prepared at postnatal days 6-8 from C57BL/6 mice or T lymphocyte-deficient nude mice (Foxn1nu/Foxn1nu, http://jaxmice.jax.org/strain/002019.html) or Sprague-Dawley rats of either sex. Slices (400 μm thick) from a McIlwain tissue chopper (Mickle Laboratory Engineering Co.) were mounted in clots of chicken plasma (Cocalico Biologicals) and thrombin (Sigma-Aldrich) on poly-L-lysine-coated glass coverslips (Electron Microscopy Sciences). Slices were incubated in roller tubes (Nunc) at 37 °C within 750 μl of NeurobasalA/B27 medium supplemented with 0.5 mM GlutaMAX and 30 μg/ml gentamicin (Invitrogen). This concentration of gentamycin was less than half the lowest concentration demonstrated to induce regenerative activity in vitro (Grøndahl and Langmoen 1993 (link)). Culture media were changed biweekly.

PFA-coated tungsten wire (bare diameter = 50.8 µm, coated diameter = 101.6 µm, A-M systems Inc.) was sterilized by 70% ethanol, affixed to the substrate of a standard tissue culture 6-well plate by silicone adhesive (4300 RTV, Bluestar Silicones, Oslo, Norway), and cured at 65 °C overnight. In each culture well, a recording electrode was fixed to the substrate with the microwire tip placed in the center and a reference electrode (same type of microwire with 1.5 cm insulation layer removed at the tip) was placed on the side (Figure 2B). The microwire-integrated culture plates were then coated with poly-d-lysine (PDL, Sigma) and incubated in a humidified atmosphere at 37 °C overnight. Plates were then washed in sterile distilled water, filled with NeurobasalA/B27 medium (Thermo Fisher Scientific, Waltham, MA, USA) and incubated for at least 3 h before the placement of hippocampal slices.

Primary culture of mouse cortical neurons was prepared as previously reported (Hilgenberg and Smith, 2007 (link)) with some modifications. Briefly, the mouse brain cortex was digested in 0.25% trypsin at 37°C for 10 min. The cell suspension was centrifuged at 1,000 rpm for 5 min. The pellet was resuspended in Neurobasal-A/B-27 medium (Thermo Fisher Scientific) with 0.25 mmol/L Glutamax-1, 40 U/mL penicillin, 40 μg/ml streptomycin, and 10% dialyzed horse serum. Cells were plated in poly-L-lysine-coated 24-well plates at 6 × 10⁵ cells per well, and cultured at 37°C in a humidified chamber containing 5% CO₂.
Transfections of MCPIP1 siRNA (4,390,771, Life Technologies) or control siRNA (4,390,843, Life Technologies) were performed as previously reported (Liang et al., 2008 (link)) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. 24 h later after 25 nM of the siRNA transfection, the TMP (10 μM) was added to the culture medium for 12 h, and the cells were subjected to OGD protocol as previously described (Gong et al., 2014 (link)). Briefly, the cells were cultured with glucose-free medium with 95% N₂, 5% CO₂ at 37°C for 120 min. The culture medium was then replaced with normal culture medium and incubated under normal conditions for 24 h. Cells without OGD were used as controls.