Isolating and Culturing Rat Retinal Müller Cells

Primary cultures of rat retinal Müller cells were prepared as previously described with modifications.^{29 (link)} The dissociated retinal tissue of rats at postnatal days 5 to 7 was seeded in culture dishes that contained Dulbecco's modified Eagle's medium supplemented with 5 mM D-glucose (normal glucose as a control). The cultures were forcibly pipetted every 3 days to purify the Müller cell population until the percentage of cultured Müller cells, which was identified by the GS expression, exceeded 98%.^{34 (link)} The cells were maintained in Dulbecco's modified Eagle's medium with 25 mM D-glucose, as a HG treatment, for 48 h to mimic diabetes in vitro. IL-17A (25 ng ml⁻¹) and/or the IKK inhibitor Wedelolactone (Wedel; 10 μM; Sigma-Aldrich, St Louis, MO, USA) were applied to the primary Müller cell cultures for 24 h in the HG condition. Ad-Act1-shRNA at a multiplicity of infection of 20 infected primary Müller cells in the HG medium, and Ad-GFP was used as a control. After infection for 24 h, the adenoviruses were removed, and the cells were cultured for an additional 24 h before harvest.