Colony counting assay also determines the toxicity of the anti-cancer therapy, but in this case it determines if the cells will grow after the cells have been treated. So the cancer cells will first be seeded on 24 well plates and incubated overnight. The next day, the cells are treated with the respective treatments and incubated. After treatment, the cells are trypsinised and replated on a new 6 well plate at a seeding density of 2000 cells per well. The cells are then maintained for 2-3 weeks with the media replaced every three to four days. At the end of the assay, the cells are stained with crystal violet for 1 hour. Lastly, the colonies are counted and imaged using a stereomicroscope (Nikon, U.S.A.) at 10× magnification, in five microscopic fields per well (one central, four peripheral).
Above is an example on how the plate will look like after the colonies have been stained. The first one would most likely be the control , hence many colonies can be seen. The last one would be the drug polymer and TRAIL, a ligand that binds to the
TRAIL receptors found on cancer cells, to induce apoptosis.
Gwendolynn Tan
TG02
0703953J
Hi Gwen!
ReplyDeleteAfter treatment, you trypsinised the cells and replated on a new 6 well plate at a seeding density of 2000 cells per well.
So, how do you determine the cells replated on the new 6 well plate are at a seeding density of 2000 cells per well?
Thks!
Cheers,
Zhang'e
0704086H
TG02
Hey Zhang'e!!
ReplyDeleteIts the same like seeding, after trypsinizing the cells, the haemocytomer is used to calculate the number of cells in 10 ul. Then calculation is done to ensure that the seeding density is 2000 cells per well, or 1000 cells per ml.
Cheers,
Gwendolynn
0703953J