Dear Researchers,
I am incredibly confused about the in vitro colony forming assay. I was asked to perform such assay to assess my cell models capability to grow without attachment to a solid substrate. Most colony forming assay protocols I have read so far only evaluate the capacity of cells to form colonies when first seeded at very low densities, which to me is only synonym of active cell proliferation. Other protocols use substrates like agar to grow cells and count colonies after 10 to 15 days. To me, this only determines cell proliferation capacity. How can I design an experiment capable of demonstrating if my cells can grow without a substrate? Should I grow cells in cell culture untreated plates?
Thank you in advance,
Andreia Peixoto
What type of cells are you talking of? For many mammalian cells, the ability to grow in soft agar is indeed a marker of a transformed phenotype, as most contact-dependent cell types need the stimulation of focal adhesion kinases for survival and proliferation. Another thing you might test is whether your cells are able to grow in suspension.
Dear Annemarie,
I’m working with adherent bladder cancer cells. Thank you for your reply. I understand now that soft agar, being semisolid, interferes with integrin-mediated substrate adhesion and focal adhesions, which leads to anoikis in most cells; thereby, the potential of a neoplastic cell to proliferate and colonize a soft agar environment is a signal of aggressive behaviour in vitro. I will try growing my cells in soft agar. Thank you very much.
You might look at this : Article The Soft Agar Colony Formation Assay
for detailed instructions (including video)- Badges
- Science topic
- Similar topics
- Microbiology
- Assays