Protocol C.6

Conditioned Medium for Retinal Explant Culture

 

 

Solutions

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)

 

Procedure

• 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.