Mouse choroid plexus epithelial cells (CPECs) were isolated by a modification of a protocol previously established for the isolation of rat CPECs [
20]. In brief, sex-matched 6–10 week old C57BL/6 mice or Immortomice
® (see below) were euthanized with CO
2, decapitated and the brain excised. For one preparation, CP was removed from 15 to 20 mice from the lateral and the 4th ventricles using a stereomicroscope. The CPs were collected, rinsed in Dulbecco’s phosphate buffered saline (DPBS, Gibco, Paisley, UK) and digested with 0.1 mg/ml pronase (Roche, Mannheim, Germany) in DPBS for 30 min at 37 °C in a shaking water-bath. The digestion was ended by addition of 5 ml DPBS without pronase. The CP clumps were re-suspended and recovered by sedimentation controlled visually by eye. The supernatant, which contained mostly single non-epithelial cells and debris, was discarded. The sedimented CP clumps were further mechanically and enzymatically disaggregated by pipetting the clumps up and down for 10 to 20 s (depending on the size of the clumps) in 1 ml of 0.025 % Trypsin–EDTA (1×) (Gibco, Paisley, UK) containing 12.5 μg/ml DNAse I (Roche, Mannheim, Germany). This procedure was followed by a sedimentation step at RT for 1–2 min and ended based on visual control of the sedimentation. The supernatant, which is enriched for single CP epithelial cells and thus more turbid, was transferred to a new tube containing 5 ml of CPEC medium (DMEM/F12 1:1 (Gibco, Paisley, UK), FBS 10 % (Gibco, Paisley, UK), 2 mM glutamine (Gibco, Paisley, UK), 50 μg/ml gentamycine (Gibco, Paisley, UK). The procedure of mechanical/enzymatic disaggregation and sedimentation was repeated 5× as this was found to yield the highest number of CP epithelial cells. The single cell suspensions were pooled and pelleted by centrifugation at 800 g for 5 min at RT and the pellet was re-suspended in complete CPEC medium, which is CPEC medium supplemented with 5 μg/ml human insulin (Sigma Aldrich, St. Louis, MO, USA), 10 ng/ml hEGF (Peprotech, Rocky Hill, NJ, USA), 2 μg/ml hydrocortisone (Sigma, Buchs, Switzerland), 5 ng/ml bFGF (Sigma, Buchs, Switzerland) and 20 μM cytosine arabinoside (Ara-C) (Sigma, St. Louis, MO, USA). To allow for further enrichment of CP epithelial cells by differential attachment to plastic, cells were resuspended in 4 ml of complete CPEC growth medium and plated on 2 non-coated petri dishes (PD35), (Becton–Dickinson Biosciences (BD Biosciences), Franklin Lakes, NJ, USA). After 2–3 h of incubation at 37 °C and 7 % CO
2, adherent fibroblasts and macrophages, characterized by their extension of cellular processes, were visible under the microscope as described [
20]. The supernatant was collected and pelleted for 5 min at 800 g. The single cell pellet was re-suspended in 0.5 ml complete CPEC growth medium and the cells were counted using a hemocytometer. 3 × 10
5 cells/cm
2 were plated either on permanox 8-well chamberslides (Thermo Fischer Scientific, Rochester, NY, USA) coated with 20 μg/ml laminin (Roche, Mannheim, Germany) for phase contrast microscopy, on laminin coated 0.33 cm
2 Transwell filters with 0.4 μm pore size (Corning, Kennebunk, ME, USA) for immunofluorescence stainings, or on laminin coated 5 μm pore size Transwell filters (Corning, Kennebunk, ME, USA) for transmigration assays. The cells were allowed to attach to the chamber slides or filter membranes at 37 °C and 7 % CO
2 for 48 h. On day 2 the cells were washed with DPBS and the medium was exchanged. The cells were grown to confluence for 1 week at 37 °C and 7 % CO
2 in complete growth medium, which was changed every other day. Continuous presence of the DNA synthesis inhibitor Ara-C specifically suppressed the growth of contaminating cells such as stromal fibroblasts. In contrast to CPECs, these cells express nucleoside transporters unable to distinguish ribose and arabinose residues and thus incorporate Ara-C into their genomic DNA, leading to the specific suppression of their growth [
30,
35].