1. It should be possible to study tumor angiogenesis with different types of multicellular tumor spheroids. Apparently the species origin of the tumor cells is not important. In our hands, tumor-induced angiogenesis could be studied with DU-145 (human) cancer cells and mouse ES cells as well as with DU-145 cells and human ES cells. Experiments were furthermore performed by the use of mouse ES cells and breast cancer cells of mouse origin (4T1).

2. Not descended from "Swiss" mice from Rockefeller Institute (probably of wild albino origin). Intensively inbred by Carworth for more than 20 generations. This line was then reduced to a single pair and progeny outbred from that point forward to form a new stock. Caesarean rederived in 1974 from a representative cross section of the Carworth CF-1 colony. Coat color Albino; carries brown behind its albino gene.

3. For this use a 1000 pL (blue tip) Gilson microliter pipet; cut the end of the blue tip with a scissor and flame it to avoid injury of the tissues.

4. Red laser light allows recording of fluorescence in deeper cells layers as compared with blue laser light because of reduced light absorption of the red wavelength.

5. The extended depth of focus option of the confocal set up generates pseudo three-dimensional image of a stack of optical sections. This pseudo three-dimensional image contains all fluorescence information of a tissue block of selected thickness (in our hands 100 pm) and can be used to calculate the area covered by immuno-labeled cell areas.

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