I am keeping my cell line for drug treatment and monitoring its effect at 24 hrs, 48 hrs and 72 hrs? Should I change the medium every 24 hrs and give fresh drug treatment or just leave the medium as it is after treatment once at the beginning? What is the difference between the two? What is the norm that is followed for cytotoxic assays such as MTT and CCK-8?
Hello Abhijit Mitra
You should not give a fresh drug treatment every 24 hours. Once the drug containing media is added to the cells, the effect of the drug is monitored at various time points (24 hours, 48 hours and 72 hours).
Subjecting the cells to fresh drug treatment every 24 hours is a wrong practice because you will disturb the cell environment as well as the interaction between the drug and the cells. Unaware of the mechanism of action of the drug, it is not known whether the drug will act within 24 hours or after 24 hours. Therefore, three time points are generally considered namely, 24, 48 and 72 hours.
The norm that is followed for cytotoxic assays such as MTT is that you should add the drug containing media only once in 96-well plates labelled as 24 hours, 48 hours and 72 hours, and incubate the plates for 24, 48 and 72 hours respectively in the CO2 incubator at 37-degree C.
After 24 hours, you may perform cytotoxicity assay (MTT) on the plate marked as 24 hours and calculate the IC50 value. Similarly, perform the MTT assay on the remaining two plates after 48 and 72 hours respectively and calculate the IC50 values for these two time points.
Best.
Dear Malcolm Nobre
what about gene and protein expression studies? Invitro these drugs are highly toxic if kept for 24hrs. Shouldn't the drug be provided for 3-4 hours following which media is changed and the RNA/Protein isolated after 24 or 48 hours?
Because even in our living biological system the drug won't always be exposed to the cells, it will be cleared from circulation and metabolized in liver
In cell line studies, it is generally recommended to change the medium and provide fresh drug every 24 hours for several reasons: 1. **Drug Stability**: Many drugs can degrade over time when exposed to light, temperature changes, or the cellular environment. Refreshing the drug ensures its efficacy. 2. **Nutrient Depletion**: Cells consume nutrients in the medium, and metabolic byproducts can accumulate. Replacing the medium helps maintain optimal growth conditions. 3. **Accurate Dosage**: Ensuring that cells are continuously exposed to the intended concentration of the drug requires replenishing it regularly. 4. **Toxicity Management**: Some drugs can become toxic over time due to their breakdown products. Changing the medium can help manage this toxicity. However, the specific protocol can depend on the drug's stability, the cell type, and the experimental design. Always consult the literature for your particular drug and cell line, and consider running a pilot experiment to optimize the treatment schedule
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