Does clumpiness matter in soft agar assay (in vitro transformation assay)? - (Oct/15/2007 )

Hi all,

I did soft agar assay with my human cancer cells (ctrl cells and oncogene-transfected cells) which are already known to form colonies on soft agar.

I did just pipett up and down and plate them. I saw really good colony formation in both but significantly more in oncogene-transfected group, which is consistent as

what we observed when we xenografted these cells in nude mice (in vivo tumorigenicity assay). Some colonies were big enough, so we could see them without

microsccope (ave 5 in ctrl, 20 in +). However when we tried same experiment again, but we could not see any visible colony even when we grow them for longer

time. The only different thing we did in 2nd experiment was "vortexing the cells" because we thought there were some clumpiness of cells when we did just "pipett

the cells" in the 1st experiment. Does clumpiness of the cells change the whole result of the soft agar assay?

FYI, we plated 100k cells/60mm dish when we got good result. We also tried 10k cells by "vortexing" but we still could not see the difference between them.

Thank you all in advance.

It is important to make single cell suspension for soft agar assay. Clumpiness will of course affect colony formation, and visualizaton of colonies. I don't think vortexing is the right way of dispersing cells. It is important to disperse cells by pipteting up and down before serum containing medium is added to stop trypanization.