In dissecting medium, cut the tail posterior to the two most recently formed somites and culture in a 40 µl drop of 75% immediately centrifuged rat serum, 25% high glucose DMEM. Inverted 35 mm dishes carrying four to six hanging drops are suspended over dishes of water in a 5% CO2 incubator at 37°C. Tails can be cultured for 20 hours, during which time 10 pairs of somites form, the normal rate of somite formation in utero. For longer periods of culture, the medium should be replaced. For best morphological development over 20 hours, tails can be cultured in rolling tube culture in 1 ml of medium.
Dissecting Medium
1. Autoclave 2.38 g HEPES in 450 ml water in a 500 ml bottle
2. Add
| Volume | Solution | Stock |
| 14.25 ml | NaCl | 29.2 g in 150 ml |
| 12.5 ml | KCl | 0.93 g in 125 ml |
| 1.8 ml | KH2PO4 | 0.68 g in 50 ml |
| 1.0 ml | MgSO4•7H20 | 1.23 g in 50 ml |
| 5 ml | Na2EDTA•2H2O | 0.019 g in 50 ml |
| 2 ml | NaHCO3 | 4.20 g in 50 ml |
| 5 ml | 100X Pen/Strep | Gibco # 15070-014 |
| 50 ml | heat-inactivated FCS | |
| 8.5 ml | CaCl2 | 1.47 g in 100 ml (or 1.68 g CaCl2•2H20) |
Mix well and store for 2 weeks or less at 4°C.
A Conlon Lab Protocol
pdf file for this protocol