I want to do a focus formation assay using mouse embryonic fibroblast which I knockdown for a gene we think is a tumor-suppressor. Should I coat the petri with something? What is the number of cells to plate when using a 6-well plate? How long should I wait before staining with crystal violet 2-3 weeks? More? I have looked at and tried different protocols but so far nothing really seems to make sense. Thanks for your help.
For anchorage-dependent colony formation assay (focus formation assay) you could plate 1,000-5,000 cells/6 well in 2 ml of media and grow them for 10-14 days before fixing and staining. Of course, cells would need to be fed during this time:) The whole idea for analyzing anchorage dependent growth is that cells are capable to attach to plast, ergo you do not need to coat plastic with anything. Otherwise, it would be anchorage-independent. Hope this helps?
Ok, thanks. That is what I was doing already... I'll add a positive control to make sure everything is fine.
When non-transformed cells grow to confluence, they stop growth (a state called cell to cell contact inhibition). However, transformed cells can override such contact inhibition of growth and therefore form foci. A focus assay is to test if a cell can form such a focus, whereas a colony formation assay is to test if a single cell can form a colony. On one hand, colonies are formed on the dishes for an anchorage dependent growth assay. On the other, colonies are formed in soft agar or in methylcellulose for an anchorage independent growth assay.
- Badges
- Science topic