Millicells cm membrane inserts

Hippocampal organotypic cultures were prepared from postnatal day 5 (P5) C57BL/6 wild-type (WT) mice (derived from the colony at the TU Braunschweig) as previously described [21 (link)]. Neonatal mice were decapitated before the hippocampi were dissected in ice-cold sterile Gey’s balanced salt solution (GBSS) and sliced transversally at a thickness of 400μm using a McIllwain tissue chopper. The slices were placed on Millicells CM membrane inserts (Millipore) and cultivated in a 37°C, 5% CO₂, 99% humidity environment in a medium containing 50% BME (Eagle, with Hanks salts without glutamine), 25% HBSS, 1ml of glucose (50%), 25% donor equine serum (HyClone), and 0.5 ml of L-glutamine (200 mM stock solution) for 100ml. To reduce the number of non-neuronal cells, a mixture of antimitotic drugs (cytosine arabinoside, uridine, and fluorodeoxyuridine; 10^–6–10^–7 M each; Sigma-Aldrich) was applied for 24h.

For whole cell patch clamp recordings, organotypic hippocampal slice cultures were prepared from postnatal day 5 (P5) C57Bl/6 mice of either sex as described previously [25 (link),26 ]. The mice were swiftly decapitated, the hippocampi were dissected and transferred into ice-cold sterile GBSS supplemented with glucose and adjusted to a pH 7.2. Transversal slices of the hippocampus were cut at a thickness of 400 µm using a McIlwain tissue chopper and let to recover for 30 min in GBSS at 4 °C. The slices were then placed on Millicells CM membrane inserts (Millipore, Merck KGaA, Darmstadt, Germany) and cultivated at 37 °C, 5% CO₂, and 99% humidity in a medium composed of 50% BME (Eagle, with Hanks salts without glutamine), 25% Hank’s Buffered Salt Solution (HBSS containing 50 mL HBSS (10×), 175 mg NaHCO₃, 147 mg CaCl₂*2H₂O, 1351 mg Glucose, total volume of 500 mL), 1% glucose, 25% donor equine serum (HyClone), and 0.5% L-glutamine. After 72 h, a mixture of antimitotic drugs (cytosine arabinoside, uridine, and fluorodeoxyuridine; stock solution of 1 mM each) was applied at a final concentration of 1 µM each to reduce the number of non-neuronal cells. After 24 h, the medium was fully changed once and then 50% of it every 7 days until the slices were used for experiments at DIV21.