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Experimental Design

Presomitic mesoderm culture
The experiments were performed on the presomitic mesoderm of the 14- stage chick embryo. The embryos were rinsed in the Ca
+2/Mg+2free Tyrode's solution (8.0 g NaCl, 0.3 g KCl, 0.05 g NaH2PO4*H2O in 1L of dH2O). The segment containing presomitic mesoderm was excised and incubated in 0.25% tripsin-0.02% EDTA for 5 minutes. The resultant mass of cells will be flushed through a narrow bore pipet to remove ectoderm, endoderm, notocord, and neural tube for at least 7 minutes. The digestion reaction was terminated with 2 ml of calf serum and spun at 1750 rpm for 7 minutes.

Treatment with FgF2 (Basic Fibriblast Growth Factor)
Twelve tissue culture dishes were coated with 1% gelatin (Sigma) in deionized water at 4oC overnight and gelatin was decanted due to melting leaving an invisible layer of gelatin. The cells were counted in 1:10 dilution using 10ml of Tripen Blue. Cells were plated at density of 2.5 * 10
4in 20 ml. Three different concentrations of FgF2 were tested:0.25 ml/10 ml, 1 ml/10ml, and 4 ml/10ml. Control was free of FgF2 and three wells per each condition were used. Two hours after plating, cells were flooded with 1.5 ml of the Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal calf serum and antibiotics containing appropriate amount of FgF2.

©Cebra-Thomas, 2000

Last Modified: 5 May 2000

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