Dear researchers,
I am trying to do colony formation assay with conditioned medium. Since the conditioned medium is collected from serum free medium, I have a question in the process:
Generally there are 3steps in the colony formation assay:
1. Preparation of base agar layer: equal volume of 1.2% agar and 2xDMEM/20%FBS for 0.6% base layer
2. Preparation of cell agar layer: equal volume of 1.2% agar, 2xDMEM/20%FBS and cell suspension for 0.4% cell agar layer
3. Quantitation of anchorage independent growth: normally adding100ul of culture medium containing cell growth activator or inhibitor to each well, examine the cell colony formation after 15days
So when I prepare the two layers of agar, should I use conditioned medium or just follow the protocol and add conditioned medium in the third step?
Thank you for taking your time to answering my question, any response will be appreciated.
Hello Lizhen,
You question is not entirely clear. If the assay is set up normally with 2x complete medium, I would set it up as usual, let the system settle, and add the conditioned medium and control spread evenly a few hours later. This set-up will allow you to titrate the effect of the conditioned medium, by using different volumes and/or diluted conditioned medium.
Good luck,
-Laszlo
If your cells for colony formation need FBS, The 20% FBS should be added to the culture. If your conditional medium is just from cell culture, the medium does not include some growth factors existed in FBS. For control, 2 times serum free medium is OK.
@ Laszlo Takacs, Dear professor, thank you so much for the answering, I edit my question to make it more clear, so do you still suggest I only use conditioned medium in the 3rd step?
@ Xin-Jiang Lu, Dear professor Lu, many thanks for the answering. I edit my question , so do you think I can only use conditioned medium in the 3rd step? Thanks for taking the time for the question.
Dear Lizhen, Yes, my suggestion is to add the conditioned medium you are testing in the 3rd step. Regards, -Laszlo Takacs
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