Preparation of Cells for Explant Coculturing

1. For culturing to assay explants, cells are grown on gelatin-coated 35-mm diameter tissue-culture dishes.

2. Starting with a just confluent flask, wash the cells with PBS, and then loosen the cells by covering with with 5 mL trypsin/EDTA (Gibco-BRL no. 610-5300AG) (store at -20°C in 5-mL aliquots) for 3 min.

3. Triturate the cell suspension thoroughly with a plastic pipet to give a complete single cell suspension. F-9 cells are tough and will tolerate much up and down pipeting.

4. Transfer the cell suspension to a 15-mL tube and add 5 mL of medium with serum. Add 40-50 drops of this suspension to 20 mL of growth medium. Put 1 mL of this suspension into each of 20 35-mm dishes. This gives a cell concentration which 5% of which will give near confluence in 2 d when incubated at 37°C.

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