I would like to understand more and its experimental methods for Colony forming assays for cancer cell line treated with a novel compound.
If you know any useful resources with regard to colony forming assay with cancer cells, pls let me know.
I would like to know 1. why is this colony forming assay carried out?
2. Some use 0.6% agarose/DMEM bottom and 0.3% agarose/DMEM upper layer with single cells suspension (2500 cells for each 6 well plate), while others are using Colony forming unit ready-made coated 96 well plates. So, do they give same results? One is on agar and another is on plastic with two different viscous medium.
3. There were researchers talking about plating cells 24 hr, then treat cells with IC50. In this case, how can we treat cells because cells are already in upper 0.3% gel.
4. Other said treat cells to certain period (hrs, days?) according to 1/2 life of compound. Then lift up cells and wash them. Then use 2 layers agarose gel as mentioned above. Is this correct method?
5. some said mixed up required number of cells, medium, gel and IC50 of compound, then use 2 layers agarose gel as mentioned and check growing of colonies. How is this method validity?
6. Are there any sterile agarose powder? One said we can just autoclave agarose and medium. In this case, I think all amino acids and valuable nutrients will be destroyed by heat. What is the best way to sterilize agarose gel solution and also to prevent destroying of nutrients in medium?
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