Millicell cm membrane insert

Organotypic hippocampal slice cultures were prepared as previously described [22 (link)]. P0 mice were decapitated, the hippocampi were dissected in ice-cold Gey’s Balanced Salt Solution (GBSS, containing: kynurenic acid (0.5 μM), adjusted to pH 7.2), sliced transversely at a thickness of 400 μm using a tissue chopper (McIllwain, Wood Dale, IL) and kept for 30 min at 4°C in GBSS. The slices were plated onto Millicell-CM membrane inserts (Millipore, Bedford, MA) and cultivated (37°C, 7% CO₂). Three days after preparation, a mixture of anti-mitotic drugs (uridine, cytosine-β-D-arabinofuranoside hydrochloride, 5-fluoro-2’-deoxyuridine) was applied for 24 h in order to reduce the number of non-neuronal cells. In addition, entorhino-hippocampal slice cultures (see [23 (link)], with minor modifications) of Thy1-GFP × APP-KO mice (P3-5) were prepared and cultivated for 12-14 days prior to imaging (age at analysis was thus equivalent to cultures prepared at P0).

Hippocampal slice cultures were performed as previously described [29 (link)]. In each independent culture, 6 male newborn rats (P2-3) from same litter of a dam were used. Briefly, after decapitation, rat brains were removed and placed in ice-cold oxygenated low sodium-containing artificial cerebral spinal fluid (containing 248 mM sucrose, 4 mM KCl, 1.25 mM NaH₂PO₄, 26.2 mM NaHCO₃, 1 mM CaCl₂, 5 mM MgCl₂, and 10 mM glucose) and then carefully placed on the platform of a tissue chopper and sliced perpendicular to its longitudinal axis using a vibrating microtome (NVSLM1, World Precision Instruments Inc.), with 400 μm thickness of each slice. Slices were transferred to Millicell CM membrane inserts (Millipore, Bedford, MA, USA) in 6-well culture plates. Each well contained 1.2 ml of pre-warmed DMEM (Invitrogen Corp., Carlsbad, CA) containing 20% horse serum (Invitrogen), 10.5 mM glucose, 12.5 mM HEPES, and 55 mM NaHCO₃ (pH 7.3–7.4). The slices were incubated in a humidified, 5% CO₂ atmosphere at 37^oC overnight, and followed by treatments with increasing concentration of dexamethasone-21-phosphate disodium salt in the presence or absence of CRHR1 antagonist antalarmin for 24h. The slices were then harvested for measurement of CXCL5 content.

Slice cultures were generated as described previously [19 (link)], with some modifications. Briefly, brains were removed from control and DSS-treated mice and placed in cold Gey's buffer solution before slicing 200 μm slices using a vibratome (Leica VT1000S). Slices were transferred to Millicell-CM membrane inserts (0.4 μm pore size, 30 mm diameter, 2–3 slices per insert; Millipore Corp) in six well tissue culture treated plates containing MEM with 25% horse serum and 6.5 mg/mL of glucose. Slices were incubated in 5% CO₂ at 37 °C for 48 h before a complete media change to MEM with 20% house serum and 6.5 mg/mL of glucose. Slices were cultured for 7 days with media changes every other day. On day 7, inserts with slices were placed in a new 6 well containing 1 mL VivoGlo luciferin (300 ug/mL) and imaged using the IVIS. Bioluminescence was quantified using Living Image software.