ORGANOTYPIC LIMB CULTURES

1. -embryos are dissected from timed-pregnant mice from 10.5 - 11.5 d.p.c.
2. -limb buds are microdissected and placed in holding medium (L15 medium supplemented with 1 x MITO+ serum extender) (all media is supplemented with 100 U penicillin/ml and 0.1 mg streptomycin/ml)
3. -limbs are allowed to sit in holding medium anywhere from 20 min to 2 hours
4. -0.2 ml of serum-free explant media (DMEM-F12 medium supplemented with 5 x MITO+ with or without 10% fetal bovine serum) is added to each well of a six-well tissue culture plate containing Cyclopore (polyethylene terphthalate; Falcon Labware) inserts
5. -limbs are transferred from the holding media onto the surface of the Cyclopore membranes (for transfer, the end of a 200 � pipette tip is removed with a sterile razor blade (to enlarge the hole), and limbs are taken up in 5 � 10 � of holding media)
6. -limbs are grown on the tissue culture inserts in a 37oC incubator under an atmosphere of 5% CO2
7. -explant media is replaced every 48 hours

Limbs can be successfully grown for 3� 4 days