Antibacterial antimycotic solution

The hippocampi for preparation of 400-μm thick organotypic slices with the preserved tissue organization were isolated from the brains of 7-day-old Wistar rats (n = 18), according to the protocol described previously [51 (link)]. The procedure was approved by the IV Local Ethics Committee on Animal Care and Use (Ministry of Science and Higher Education). The slices, obtained by cutting the chilled hippocampi by use of a McIlwain apparatus, were placed on Millicell-CM (Millipore) membranes and cultured initially in DMEM medium (Gibco) containing the following supplements: 25% horse serum (Gibco), 25% HBSS (Gibco), 2 mmol/l L glucose (Sigma), 5-mg/ml HEPES (Gibco), B27 supplement (Gibco), and an antibacterial–antimycotic solution (Sigma). On the day following establishing the ex vivo culture, the serum concentration in the culture medium was gradually decreased and therefore from the 5 day in vitro (DIV) onwards, the slices were kept for the following 7 days in serum-free media and in normoxic gas conditions (5% O₂, 5% CO₂).

All solvents were HPLC grade and were degassed and filtrated before their application, whereas all other chemicals were of analytical reagent grade. Dulbecco’s modified eagle medium (DMEM), GlutaMAX™, was purchased from Life Technologies, Paisley, UK, while antibacterial/antimycotic solution, phosphate-buffered saline, trypsin-EDTA, glucose oxidase/peroxidase and o-dianisidine, baker’s yeast α-glycosidase, p-Nitrophenyl-α-glucopyranoside sucrase, maltase, lactase, and acarbose were obtained from Sigma-Aldrich, Pool, UK, as well as the following reagents: ethanol, hexan, formic acid, and methanol. The multi-well Tecan’s Spark® plate reader was obtained from Tecan Group Ltd., Mannedorf, Switzerland.