Choroid culture protocol Autoclave preparation: PBS, water, scissors, spatula, forceps, pipette tips, beakers, bottle for medium. Under hood in Cell Culture Room: Prepare choroid culture medium DMEM 100U/ml Penicillin 100ug/ml Streptomycin 0.25ug/ml Amphotericin B If required: Phenol red = 3.18ul/ml Glutamine = 20ul/ml 96 well tissue culture plate typically used Pipette out 100ul medium per well. Prepare label plan of chick number, eye, treatment and well for 96 well plate. Prepare Ringers solution required for storage of enucleated eyes In Chick ante room: Need: Large beaker lined with bag for chick body Guillotine Scissors Forceps to dissect out eyeball Vials containing Ringers solution to collect eyeballs (labeled for each eye) Large beaker containing ice to keep vials Sacrifice, enucleate & immerse eyeball in Ringers solution on ice Under hood in Cell Culture Room: Need: Sterile Petri dishes + fine dissection instruments Base plate 8 mm punch, 70% alcohol Time tape Sterile Razor blades Paint brush Vials for transferring eyes Procedure: Place paint brush and baseplate wax in alcohol to sterilize. Label petri dish with eye number Use razor to nick and scissors to make circumferential cut around equator of eyeball Remove iris, vitreous Flush with PBS to remove adhering tissue Place eyecup on the baseplate Use Trephine/punch to punch out tissue button (8mm) at a tangent to the pecten. (Avoid pecten as this is an area where the choroid and RPE are difficult to separate*). Use brush to remove retina and RPE from the punch, flush choir/sclera punch with PBS Hold the edge of the punch firmly to the petri dish with forceps. Place the spatula at the edge and use it to peel off the choroid from the sclera. Place the choroid in the well of the microplate. Cover and grade quality of the dissection under a microscope. Incubate for 24 hrs and collect medium. *Eyecup is placed on a piece of Truwax® Baseplate Wax before punching. Once the punch is made, move it off the wax into petri dish; hold punch down by edge using forceps and used a flat spatula to scrape off the choroid from the sclera. No need to stick the sclera down.