Transwell clear polyester membrane

Primary neurons were isolated as previously described and were seeded on 24 mm inserts with 0.4 µm pore size (Transwell-Clear Polyester Membrane; Corning Inc., Corning, NY, USA) at a density of 0.5·106 cells with pre-suit corresponding medium, and incubated for 6 days. Then, IGF-I-treated differentiated OLN-93 cells were seeded below the neurons. Samples from each of the treatment groups were collected for protein analysis after co-culture for 3 days. For immunofluorescence staining, IGF-I-differentiated cells were also plated on PDL-C-coated 14 mm slides and co-cultured for 3 days with each of the neuron treatment groups.

For TER measurements, the cells were seeded at a density of 10⁵ cells on cell culture inserts (Transwell, clear polyester membrane, 12 mm diameter, 1.12 cm² area, 0.4 μm pore size; Corning B.V., Schiphol-Rijk, the Netherlands). The inserts for IPEC-J2 cells were coated with rat tail collagen type I (Serva Electrophoresis GmbH, Heidelberg, Germany). TER was measured by a Millicell-ERS (Electrical Resistance System; Millipore GmbH, Schwalbach, Germany). TER values were measured every two hours and corrected for the resistance of blank filters and for the membrane area.