Pen strep l glutamine

ARPE-19 human retinal pigmented epithelial (RPE) cells were obtained from American Type Culture Collection. Culture media was composed of equal parts F12 and DMEM (GE Life Sciences, Pittsburgh, PA), supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 20 mM HEPES (Sigma-Aldrich, St. Louis, MO), and 1% Pen/Strep/L-glutamine (100 units/mL, 100 μg/mL, and 300 μg/mL respectively) (GE Life Sciences, St. Louis, MO). Cells were cultured at 37° C with 5% CO₂ and seeded onto acid-washed coverslips in 6 well plates (Corning). Cells were transfected approximately 24 hours following seeding using 1 μg plasmid DNA for each receptor and 3 μg FuGENE transfection reagent per μg plasmid (Promega, Madison, WI) in transfection media. Transfection media was identical to culture media, except for the fact that it lacked phenol red and Pen/Strep/L-glutamine. These ingredients were omitted to increase transfection efficiency.

As described previously, ARPE-19 human retinal pigmented epithelial (RPE) cells were obtained from American Type Culture Collection²⁸ . Cells were cultured in media containing equal parts F12 and DMEM (GE Life Sciences, Pittsburgh, PA). Media was supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 20 mM HEPES (Sigma-Aldrich, St. Louis, MO), and 1% Pen/Strep/L-glutamine (100 units/mL, 100 μg/mL, and 300 μg/mL respectively) (GE Life Sciences, St. Louis, MO). Cells were cultured at 37° C with 5% CO2. For imaging experiments, cells were seeded onto acid-washed coverslips in 6 well plates (Corning). Approximately 24 hours after seeding, cells were transfected using 1 μg plasmid DNA for each receptor and 3 μg FuGENE transfection reagent per μg plasmid (Promega, Madison, WI) in transfection media. Transfection media was identical to culture media, except for the fact that it lacked phenol red and Pen/Strep/L-glutamine to increase transfection efficiency.