I want to do colony formation assay using U-87 MG cells, human brain astrocyte tumor. But I have no idea about that. What should I pay attention to?
You should seed 100-1000 cells per 6cm petri dish. It depends on the cell size, population doubling and how malignant your cells are. Larger, faster dividing and more malignant cells can be seeded in lower density. For example I am seeding MDA-MB-231 breast cancer cell in density 625 cells/petri dish. Then you are waiting approximately 2 weeks, changing the growth medium once, twice a week. Then you stain the cells with crystal violet, count the colonies. You must remember that a colony is a cluster of cells consisting fo more than 50 cells. Good luck :)
Both of these assays were performed using U87 cells. In order to assess the self-renewing capacity, i'd also try the sphere forming assay, which also works fine for U87 as an alternative.
Colony formation:
To assess the tumor colony forming capacity of the cells, 500 cells per 6 well plate in triplicate were seeded in the growth medium. To allow the single cells to form colonies, the cells were cultured for 2 weeks and every 2 to 3 days the medium (DMEM, 10% FBS) was changed. After washing the cells twice with PBS, the crystal violet staining (0.5% Crystal violet (Fluka #61135) in 20% Methanol/H2O, sterile-filtered through a 0,45 μm PDVF filter; stored at +4°C) was performed for 5 to 10 minutes. Subsequently, the cells were washed again twice with PBS and colony numbers were counted.
Sphere formation assay
To determine the sphere formation capacity (sphere forming units (SFU)), cells were split and 6 wells per condition were seeded in tumor sphere medium to suspension 6 well plates (primary glioblastoma cells, 1000 cells per well) or pHEMA coated 6 well tissue culture plates (glioblastoma cell lines, 500 cells per well (U87)). Spheres consisting of 4 or more cells were counted and presented as the percentage of sphere forming cells after 7 days.
Tumor sphere medium:
500 ml DMEM/F12, 10ml B27 serum-free supplement minus vitamin A (#12587-010, Gibco), 1 ml amphotericin B (#A2942, Sigma-Aldrich), 2.5 ml 1M HEPES (#1563-056, Gibco), 0.5 ml gentamicin (50 mg/ml) (15750-045, Gibco) and add human recombinant EGF (final concentration 20 ng/ml) (#AF-100-15, PeproTech), human recombinant bFGF (final concentration 20 ng/ml) (#100-18B, PeproTech) to the dishes freshly.
Hope that helps.
Well, i have no explanation for that. Did you seed the 3000 cells in 6wells or 10cm dishes? I never had a problem with 500 cells in 6wells with 2 ml of DMEM/10%FBS w/o antibiotics. Did you check cell viability before seeding? From my experience 3000 cells are a lot, as you want to obtain single colonies which you still should be able to count properly afterwards.
Seed 5000 cells per well in 6 well plate and be patient wait for 7 to 15 days.
it will work.
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