Conditioned Medium for Retinal Explant Culture

Monday, May 03, 2004

Description
Conditioned Medium for Retinal Explant Culture

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癈 in 240 microliter aliquotes.

?To use add 20 microliters per explant each day.

Recipes
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)

Supplies

Tips

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Conditioned Medium for Retinal Explant Culture