4.2. Cell Culture Methods

661W cells were obtained from Dr. Muayyad Al-Ubaidi, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA, under terms of a Material Transfer Agreement. 661W cells are a simian virus 40 (SV40) large T antigen-immortalized cell line derived from a benign, induced retinal tumor isolated from an early postnatal mouse, and had been originally demonstrated to possess properties of cone photoreceptors [100,226]. Protocols and techniques for expanding initial 661W cultures and cryopreserving source stocks, for subculturing and adaptation to a more defined medium, and for the extensive characterization and authentication of these cells in our laboratory using genomic, transcriptomic, immunochemical/immuno-histochemical, and morphological analyses and criteria, have been previously provided in detail [21,242], and are briefly summarized here. Cells were received in passage 24, and underwent gradual adaptation during the next several passages from medium containing 10% fetal bovine serum (Atlanta Biological, Atlanta, GA) to growth medium with the following attributes (see also Table 3 in [242]): a greatly reduced serum component (0.2% (v/v) bovine calf serum (BCS; Lonza, Walkersville, MD, USA)); formulation using a basal medium composed of 1:1 DMEM:F-12 (both HEPES modification; Sigma-Aldrich); addition of supplements based on an updated recipe for Neurobasal medium [243,244]; and containing a HEPES-buffered saline extract of a bovine retinal homogenate as the only other undefined component, contributing added total protein for a final concentration of 6 mg per liter of complete medium. Cells were subcultured using Accutase [245] for enzymatic release from the growth substrate.