In my study, on anticancer activity of a drug, I found that IC50 dosage for 2 cell lines are different. In one of my cell lines, the concentration is 60 uM for 24 h and for the other it is 60 uM for 48 h. So which concentration and time should I use to find out the molecular mechanisms such as pathway regulation and other processes? Can I use the same 60 uM for 24 hours, followed by whole cell lysate preparation analysis, or do I have to go for some other dosage and some other time period for signalling molecule analysis? Does the death of 50% of cells have any impact on the expression of loading control proteins like beta-actin?
1. Every cell line is expected to have its own response to a given drug. Dont be surprised at all.
2. 50% cell death is not good at all. This means apoptosis and autophagy is too far gone so that non-specific protein loss is a big concern. beta-actin can still be intact in these cases.
3. Since SPECIFIC signaling is expected to occur well before full death occurs, you can try treating cells up to 12 hrs and looking at signaling. I would do 4hrs, 8hrs and 12 hrs on the cell line that responds in 24 hrs.
Good luck!
Hi Pramod,
According to me, Since you got different IC50 for different cell lines, to find the pathways effected by your drugs its best to chose one cell line as a model. Drug concentrations examined if necessarily kept at physiological levels would be great (I know you can increase the concentrations invitro but I prefer physiological levels). After identifying the pathways in one cell line you can examine the same in the other cell line as well (may be with varying concentrations of your drug). I don't think you would have any significant alteration in beta-actin.
1. Every cell line is expected to have its own response to a given drug. Dont be surprised at all.
2. 50% cell death is not good at all. This means apoptosis and autophagy is too far gone so that non-specific protein loss is a big concern. beta-actin can still be intact in these cases.
3. Since SPECIFIC signaling is expected to occur well before full death occurs, you can try treating cells up to 12 hrs and looking at signaling. I would do 4hrs, 8hrs and 12 hrs on the cell line that responds in 24 hrs.
Good luck!
have u tried using the same concentration at 36 hrs?
as what others have mentioned, some drugs have different reactions towards cell lines.
The method to determine this is not using in-vitro but by in-vivo, half-life and ld50
as in-vivo and in-vitro shows different results.
Thank you Prem.... Now am planning to check the signalling sequences at different time points....
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