I am experimenting with cancer and non-cancer cells in a 12-well plate for 4 days with a seeding density 1*10^4/well, however, I noticed that the control group growth rate slows down on D3. Should I consider changing the medium on D2 in all groups and adding the drug once more given that the total volume/ well is 3 ml?
Hello Nagwan Elsayed
No, you should not change the medium during drug treatment. Once you add the media containing the drug, it should be left untouched for 96 hours. Changing the medium on D2 will disturb the cellular environment as well as the interaction between the drug and the cells.
Most of the time, the mechanism of action of the drug in unknown. So, whether the drug will act within 24 hours or after 24 hours is not known. I would recommend the use of different time points namely, 24hrs, 48hrs, 72hrs and 96 hrs. unless you have optimized the period of incubation.
Changing the medium on D2 may not be the solution to the problem. There may be some other reason for slow growth rate on D3 in the control group. You may consider a few of these provided below.
1. There may be variation in temperature when the culture vessels are stacked, the lowest vessel being closest to the metal shelf will heat up faster. So, avoid stacking of culture vessels. Also, do not open the incubator door frequently.
2. Evaporation affects cell growth rates and patterns. Minimize it by keeping water reservoirs in the incubator full.
3. Vibrations can cause unusual cell growth. These vibrations can come from a loose fan motor in the incubator or from motorized appliances. Keep incubators on sturdy surfaces that don't have any other pieces of equipment that vibrate.
4. Uneven handling or insufficient mixing of cell culture media can cause foam or bubbles, which can hinder cell growth and attachment. Pipette media carefully to ensure bubble-free culture environment.
5. Watch out for contamination, especially mycoplasma contamination.
Best.
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