I am working on the 4T1 murine breast cancer cell line. from the first day I bought this cell line, the cell's growth rate is extremely high. I intend to make these cells resistant to a single drug. It is required to expose the cells to the concentration of IC20 of the drug for a few days.
My problem is when I treat the cells with the drug at the confluency of 80 percent after two days, the cells' confluency surpass 100 percent, and the cells undergo the death phase. I am wondering what I can do about this problem?
I appreciate any suggestions.
PS: HI-FBS is being used for cell culture (8 percent).
PS2: cells are being seeded at 30 percent, and the cells do not proliferate when seeded at less than 30 percent.
PS3: in available protocols for generating drug-resistant cell lines, it is suggested that it is better not to add antibiotics.
PS4: I am using T25 flasks.
Shervin Afzali One option to consider is the use of mitomycin C - as this is an antibiotic, you could induce cell cycle arrest with a overnight incubation, then replenish the media and hope that your cells have a reduced proliferative potential
Article Mitomycin C as an alternative to irradiation to inhibit the ...
i disagree here, as MitC is a DNA cross-linker, you will completely change the dynamics in your culture, and this will impact the results when you will test the efficacy of a drug. Each culture has 1-3% of "stem-like" cells on average, while other cancer cells are more differentiated. with MitC you will kill them (slowly). Rather, reduce the serum% to 1-2% or even 0.5%. Some cancer lines can grow without serum. This will help you to have slower growth. Try to gradually "teach" the cells to adjust to lower serum %, dont shock them with 1% if you are growing in 8% (btw, it is way too high for most cancer cell lines). Hope it helps
Hi Shervin
I was wondering which drug you've selected to induce resistance in this cell line?
Do you provide the cells with a fresh dose of the drug after 24hours? Multiple doses may prevent your issue.
Considering that the IC20 leads to overconfluency within 48hrs, perhaps you could induce resistance using a higher drug concentration based on MTT results (i.e.: IC30-IC80).
I hope this helps!
-Taskeen
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