Cells that contain a transforming oncogene will grow without contact inhibition and on a
confluent monolayer of non-transformed cells will form dense, raised foci which can be
visualized by fixing and staining the cells.
Transfect cells according to Tissue Culture - 9 and 10 but do not change medium to that containing selection antibiotic, maintain in medium + serum + pen-strep only.
Continue to change the medium every 3 to 4 days for 3 weeks.
Stain the dishes with Geimsa stain, see below.
Testing established cell lines
Trypsinise the test cell line and a dish of untransformed Rat 2 cells (Tissue Culture - 5), count both cell suspensions (Tissue Culture - 6).
Plate out 102 test cells with 5 x 105 Rat 2 cells onto a 10cm dish in non- selective medium.
Change the medium every 3 to 4 days for 3 weeks.
It is very important to include both a positive and negative control with these assays.
A crude value for transformation efficiency can be determined by counting the number
of foci obtained as a percentage of the number of cells plated.
Staining foci with Geimsa stain
Rinse plates with PBS.
Add 5ml/plate of 10% formaldehyde in PBS, leave at room temperature 30 min.