Conditioned Medium for Retinal Explant Culture
Explant Culture Medium
45% HAMS F-12 (Gibco #11765-054)
45% DME (Gibco #10313-021)
10% FCS (HyClone)
Insulin (5 mg/ml 1000X stock in H2O with HCl)
(10 ml/liter) L-glutamine (200 mM, Cellgro # 25-005-CI)
(10 ml/liter) Penn/Strep (Cellgro #30-002-CI)
(10 ml/liter) HEPES (Cellgro #25-060-CI)
Remove E14.5 rat retinae with lens intact in prewarmed explant culture medium. Immediately place 4 retinae on a polycarbonate filter (1 micron pore size, 13 mm diameter, Corning/Nucleopore #110410) in 1 ml prewarmed explant culture medium in a 12 well dish.
Culture for 4 days and each day add as much medium as possible (50-100ml) to each explant (4 retinae) from the well containing the explant.
On the fourth day, remove the explants, dilute the cultured supernatant twofold with fresh explant culture medium, and filter through a syring filter that is low protein binding such as Millex-GV (0.22 micron pore size).
Store at -80°C in 240 microliter aliquotes.
To use add 20 microliters per explant each day.