As shown in Supplementary Materials Video S1, care was taken to minimize movement over the ITO songs and to prevent focusing the laser directly upon the MEA electrodes, as these actions could damage the electrodes and insulation. for the design and maintenance of custom neural circuits for functional analysis. Tergazyme (Sigma Aldrich, Cat# Z273287, St Louis, MO) answer, then washed three times with DI H2O, and finally sterilized under UV, in a biosafety hood, for 1 h. Poly (2-hydroxyethyl methacrylate) (p-HEMA) GS-9620 (Sigma Cat#3932) answer was prepared as previously explained, by dissolving 20 mg/mL of p-HEMA in 95% ethanol (EtOH), overnight, rocking at RT [9,10]. A p-HEMA covering was necessary to prevent adhesion of retinal cells to some areas of the MEA so that the optical tweezers could pick up and move the retinal cells. This answer was applied to specific areas of the surface of the MEA by placing the MEA in a 35 mm dish, such that it was inclined at a 60 angle. Then, 100 L of p-HEMA answer was cautiously dripped onto the surface of the MEA, being careful to not allow the p-HEMA treatment for cover the Rabbit Polyclonal to MRPL21 central electrode region (Physique 1). The MEAs were then laid smooth into 94 mm dishes, covered, and allowed to dry for 1 h in a biosafety hood. The MEA was then rotated 90, and the p-HEMA covering was repeated so that ? of the total surface area of the MEA was coated, but not the electrodes in the center (Physique 1). If p-HEMA did accidentally drip onto the electrode area, the MEA was quickly sprayed with 70% EtOH, washed 3 times with sterile GS-9620 DI H2O, and allowed to dry. The MEA was then recoated with p-HEMA, using the actions previously explained. Open in a separate window Physique 1 Procedure for covering MEAs with p-HEMA. (1) MEA is usually balanced at a 60 angle in a 35 mm dish, and 100 L of p-HEMA is usually dripped onto the MEA, taking care to not allow the electrodes in the center of the MEA to be covered with p-HEMA. The MEA is usually then covered in a 90 mm dish and allowed to dry for 1 h. (2) The MEA is usually turned 90, and p-HEMA is usually again placed at a 60 angle in a 35 mm dish, and 100 L of p-HEMA is usually dripped onto the MEA, again taking care not to allow p-HEMA to drip onto the center electrodes. The MEA is usually once again covered in a 90 mm dish and allowed to dry for 1 h. (3) A PDMS ring is usually applied to the MEA, and Vaseline is usually applied round the ring, to prevent leakage of media. GS-9620 (4) The MEA is usually coated with 75 L of Sal-1. (The black bar is usually provided to help visualize changes in orientation.). Polydimethyl siloxane (PDMS) rings were made to hold liquid around the MEA. PDMS (Dow Corning Corporation Cat#3097358-1004) was made as previously explained . Briefly, elastomer base was vigorously mixed with the curing agent in a 10:1 ratio by weight. The solution was then placed in a desiccator, under vacuum, for 30 min, to remove air flow bubbles. The PDMS polymer was poured into a 94 mm culture dish, placed under vacuum for another 30 min, and finally cured in a 70 C oven for at least 2 h. A ring with a 1 outer and ? inner diameter was punched from your PDMS slab, cleaned using Scotch Tape, and sterilized by submerging.