I'm planning to perform a clonogenic assay in agar to evaluate the tumor suppressor effect of a gene I'm studying. Going through protocols I see different agar concentrations for top and bottom layers in Petri plates. Can somebody help me in understanding how to determine this parameter?
Moreover in many protocols a colony is defined to consist of at least 50 cells. When cells grow as a layer it is easy to count them but how is it possible when colonies are spheres?
Hi Gianluca,
usually one would have to test the right agarose concentration of the cell-containing layer, but most transformed cells should be able to grow in 0,2%-0,3% agarose. I always used 0,3% since lower concentrations are problematic for downstream actions such as staining.
The concentration of the bottom layer (usually 0,6%) isn't that important, since it serves as a barrier between the cell-containing layer and the cell culture plates. If cells get in contact with the cell culture plates they would start to grow below the agarose as a monolayer, which you don't want.
More important is that you use the right low-melting agarose to avoid toxic effect from the agarose. Seeding single cells is also a mayor point, since you want to analyse anchorage-independent-growth of single cells.
The cell number of colonies are usually just approximations I guess. The most important point is that you get clonal colonies that exhibit continous growth and that don't count colonies that stopped proliferating after a view cell divisions.
If you really wan't to find out how many cells are in a colony of a certain size, you could try picking colonies out of the agarose, trypsinize and resuspend in a very low volume and could them in a counting chamber.
Best,
Kai
We have carried out the colony assay in our recent manuscript. And I would suggest to look at it. Here is the reference:
Effect of bortezomib in combination with cisplatin and 5‑fluorouracil on 4T1 breast cancer cells, MOLECULAR MEDICINE REPORTS 8: 277-281, 2013.
If you have any questions, please do not hesitate to contact.
Hi Gianluca,
usually one would have to test the right agarose concentration of the cell-containing layer, but most transformed cells should be able to grow in 0,2%-0,3% agarose. I always used 0,3% since lower concentrations are problematic for downstream actions such as staining.
The concentration of the bottom layer (usually 0,6%) isn't that important, since it serves as a barrier between the cell-containing layer and the cell culture plates. If cells get in contact with the cell culture plates they would start to grow below the agarose as a monolayer, which you don't want.
More important is that you use the right low-melting agarose to avoid toxic effect from the agarose. Seeding single cells is also a mayor point, since you want to analyse anchorage-independent-growth of single cells.
The cell number of colonies are usually just approximations I guess. The most important point is that you get clonal colonies that exhibit continous growth and that don't count colonies that stopped proliferating after a view cell divisions.
If you really wan't to find out how many cells are in a colony of a certain size, you could try picking colonies out of the agarose, trypsinize and resuspend in a very low volume and could them in a counting chamber.
Best,
Kai
I did the colony assay about 10 years ago. I look at my old protocol files and found the detail method. I am attaching here and hopefully it will help.
A. Bone marrow cells preparation:
1. Flush the marrow cells from tibia and femur bones with IMDM containing 2 % FBS.
2. Wash the cells for 3 times.( 2000 rpm for 10 min at room temp).
3. Resuspened the cells with the same buffer into 3-4 ml.
4. Cell counting: take 10 ul cells into 90 ul PBS and then add 10 ul saponin and wait for 1 min. Add 90 ul PBS right away. This is 20 times dilution.
B. Seeding the bone marrow cells on 6 well plate:
1. Adjust the cell concentration at 3 ×106 /ml.
2. Add 20 ml IMDM and antibiotics (amp/sp) into the methycellulose (catalogue # 03231). Shake gently and wait for the bubble up to the top.
3. Add 1/10 volume cells and GCSF (final conc: 10 ng/ml) into methylcellulose. Inverting shake 50 times and no stop.
4. Seed 2 ml of mixture onto the 6-well plate and the cell conc will be 4 ×106 per well.
5. Put in the 5 % CO2 incubator and wait for 7-8 days.
I was using Methylcellulose instead of agarose in CFC assay before, the advantage are the available of multiple commercial products (without specific cytokine added) and to easily plucking out colony using a p200 pipette. These vendors have details protocols and pictures of colony on their website that will be helpful and to give you guidelines so that you can adjust the protocol to best fit your needs.
As said by others, the determination of a proliferating colony isn't difficult when you see one. 50 is only a rough number and as you monitor daily or every two days you will not miss any proliferating colony. The time you start seeing the colony to develop depends on the cell type to use, it could be up to 7-8 days.
Do multiple dilutions when you seed the cells, so that you will neither have overgrown colonies nor under-grown colonies.
Hi, you have already got very good suggestions and protocols, I just want to highlight a commercial kit from Cellbiolabs (catalog number CBA-140 or CBA-130) I used a few years ago, which was a very good material to start with. I would also suggest you to include a positive control in your experiments, for example the cell line A549, which is able to form nice colonies in soft agar. Good luck!
The "50 cells per colony" is a measure of clonogenicity, the cell and its progeny have undergone between 5-6 divisions. The suggestions already made here about agar density are excellent. When I moved from looking at clonogenic survival (not done in agar) to this assay I changed from the standard of "50 cells per colony" to measuring colonies >10 microns. However your colony size will probably depend on the time you allow the cells to grow and their normal division rate.
Good luck.
HI, I also used methylcellulose method for B cell colony isolation. You get the flexibility of buying either plain one or supplememented ones with different cytokines to aid colony formation for your cell type. You can seed the cells and 3 days later you can start monitoring the colonies, usually depending on how fast growers your cells are. Usually start with colonies of about >50 cells. You can collect these individual colonies by cutting the tip of a 200µl pipette tip, resuspend them in culture medium and do a cell count or transfer them into another vessel to expand them. You can also use the CFSE method, you add the dye to your live cells before seeding and after 7 days you measure the dye intensity with a flow cytometer. As the cells divide the dye intensity is reducing, for every cell division the dye intensity halves. Make sure you monitor your cultures daily and avoid any satelite colonies, try and pick the more isolated ones.
good luck
Hi Gianluca, Am I right in assuming that you are going to take your tumour cells and plate them in agar and count colonies and in parallel transfect your tumour supressor gene and see how the number of colonies is reduced? Just a few points, are you using a stable transfectant(s) or are you going to do a transient transfection/infection? It is just that it should take a few weeks for the cells to grow and you might find that cells may have kicked out the plasmid if you transfect, so you may be better of with a number of stable transfectants which express your gene at different levels, and you could then correlate expression with function. I would also point out that a 50 cell colony is just visible to the naked eye.
good luck!
Andy
HI, here is the overview of blebbishield mediated colony formation, which should be ideal to test tumor suppressor activity
https://www.researchgate.net/publication/297816983_Blebbishield_emergency_program_an_apoptotic_route_to_cellular_transformation
The following papers show how LiCl2+Cisplatin can be used to test colony formation after apoptosis (low yield but larger colonies)
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
https://www.researchgate.net/publication/291526211_Endocytosis_and_serpentine_filopodia_drive_blebbishield-mediated_resurrection_of_apoptotic_cancer_stem_cells
and the following paper shows how cycloheximide can be used to test colony formation after apoptosis (High yield but smaller colony size)
https://www.researchgate.net/publication/292615481_Mitochondrial_oligomers_boost_glycolysis_in_cancer_stem_cells_to_facilitate_blebbishield-mediated_transformation_after_apoptosis
Good Luck...!!
Article Blebbishield emergency program: an apoptotic route to cellul...
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
Article Endocytosis and serpentine filopodia drive blebbishield-medi...
Article Mitochondrial oligomers boost glycolysis in cancer stem cell...
A criterion of 50 or more cells to be considered as a surviving clonogenic colony comes from the following pioneering radiation biology paper.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2136626/pdf/653.pdf
Even with the same cell number, the size of colonies are different, e.g., either when tightly packed (densely stained), or when loosely packed (lightly stained).
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