USC Organotypic Slice Culture Protocol
Organotypic slice cultures.
Adult and embryonic brain tissue was sliced at a thickness of 400 µm (Leica VT1200S) in ice-cold artificial cerebral spinal fluid (aCSF; 124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 24 mM NaHCO3, 5 mM HEPES, 12.5 mM glucose, 2 mM MgSO4, 2 mM CaCl2). Slices were immediately placed directly on top of culture inserts (Millipore Millicell inserts PITP01250) in a 6-well culture plate containing 1 ml of culture media. Embryonic slice cultures were maintained in Dulbecco's modified eagle's medium (DMEM; Life Technologies) containing 2 mM GlutaMAX, 44 mM NaHCO3, 25 mM Dglucose, 1 mM sodium pyruvate, 25 mM HEPES, 10% fetal bovine serum, 1% penicillin/streptomycin. Adult slice cultures were maintained in DMEM (Life Technologies) containing 2 mM GlutaMAX, 44 mM NaHCO3, 25 mM D-glucose, 1 mM sodium pyruvate, 25 mM HEPES, 10% horse serum, 5% fetal bovine serum, and 1% penicillin/streptomycin. Slices underwent a 50% medium exchange every 2 days and were kept in a cell culture incubator set at 37°C with 5% CO2.