Materials and Reagents

1. Target cell lines can be those with and without drug treatment or expressing the gene of interest.
   Note: Our example includes four cell lines that are engineered to have inducible expression of tumor suppressor gene and the corresponding vector alone controls.
   
2. Growth factor-reduced BD Matrigel (BD Biosciences, catalog number: 354230 )
   
3. Dulbecco’s Modified Eagle Medium (DMEM) (High Glucose L-Glutamine, 500 ml) (Life Technologies, Invitrogen™, catalog number: 11965-092 )
   
4. Fetal bovine serum (Life Technologies, Invitrogen™, catalog number: 26140-079 )
   
5. Primary Human Umbilical Vein Endothelial Cell (HUVEC) (Life Technologies, Invitrogen™, catalog number: C-003-5C )
   
6. Medium 200PRF (Life Technologies, Invitrogen™, catalog number: M-200PRF-500 )
   
7. Low serum growth supplement (LSGS) (Life Technologies, Invitrogen™, catalog number: S-003-10 )
   
8. Conditioned medium (CM)
   

Equipment

1. Tissue culture setup
   
2. Inverted microscope with digital camera (Nikon TMS)
   
3. Scion Image software downloaded from the NIH website
   
4. 96-well plates
   
5. Centrifuge
   
6. Cell counter
   
7. T75 tissue culture flask
   

Procedure