How to make an immortalized cell line/ How to determine IC50? - Protocol for developing a drug resistant cell line? (Jan/29/2007 )
Hi all,
I am trying to study for chemotherapy resistance for two drugs. I was wondering if anyone knew the exact protocol for developing a drug resistant cell line. From the articles that I read they only say that drug resistant cell lines were produced from stepwise additions of the drug of interest but there was no detailed protocol. At what confluency is the drug initially added? Do we increase the drug dosage after every day and would we double the concentration each time? Also, I'm not sure how to determine the IC50 values for the cell lines. I did the MTT cell proliferation assay using different drug dilutions and obtained absorbance values from ELISA readings. However, I'm not quite sure what to make of the values in order to calculate the IC50. I have the values for untreated and drug treated cells but I don't think that half the absorbance of the control equate to the IC50 does it? Anyone have any idea?
I am trying to study for chemotherapy resistance for two drugs. I was wondering if anyone knew the exact protocol for developing a drug resistant cell line. From the articles that I read they only say that drug resistant cell lines were produced from stepwise additions of the drug of interest but there was no detailed protocol. At what confluency is the drug initially added? Do we increase the drug dosage after every day and would we double the concentration each time? Also, I'm not sure how to determine the IC50 values for the cell lines. I did the MTT cell proliferation assay using different drug dilutions and obtained absorbance values from ELISA readings. However, I'm not quite sure what to make of the values in order to calculate the IC50. I have the values for untreated and drug treated cells but I don't think that half the absorbance of the control equate to the IC50 does it? Anyone have any idea?
there are some questions in your post; I may refer to the IC50: It only makes sense if there is a function or activity (f.i. of enzymes, transporters, ATPases etc) which are inhibited by your drug; what do you like to measure? MTT measure cell viability which is useful in terms of determination of LD50
for selection of a drug resistant cell there may be no standard protocol as different cells react differently to a certain drug; your ideas go in the right direction
moreover, a drug resistant cell line is not necessarily immortal
I was trying to determine the concentration of drug needed in order to inhibit cell growth by 50%, which is the IC50. However, I'm not sure how to determine that by looking at the absorbance values. How do I know which concentration actually decreased the cell growth by 50%? How exactly do the absorbance values correspond to the percentage of growth, is there a formula for this that can be calculated?
cell growth in the sense of cell division? so, you may determine which cell cycle arrest (G0/G1 or G2) occurs in dependence of your drug; with MTT you cannot distinguish between,on the one hand, cell cycle arrest and, on the other hand, non-cell cycle arrest but increased apoptosis or necrosis; you have to use various tests (FACS, MTT, SRB, apoptosis and necrosis assays)
actually, it should be determined what the target of the drug is
cell growth in the sense of cell division? so, you may determine which cell cycle arrest (G0/G1 or G2) occurs in dependence of your drug; with MTT you cannot distinguish between,on the one hand, cell cycle arrest and, on the other hand, non-cell cycle arrest but increased apoptosis or necrosis; you have to use various tests (FACS, MTT, SRB, apoptosis and necrosis assays)
actually, it should be determined what the target of the drug is
O i see,
That's sounds much more complicated. I thought there was a way to determine IC50 from MTT. Maybe the halfway point between the absorbance of positive control (cells with no drug treatment) and negative control (absorbance with media alone)...and the absorbance at half point should give me the concentration of drug at IC50?
cell growth in the sense of cell division? so, you may determine which cell cycle arrest (G0/G1 or G2) occurs in dependence of your drug; with MTT you cannot distinguish between,on the one hand, cell cycle arrest and, on the other hand, non-cell cycle arrest but increased apoptosis or necrosis; you have to use various tests (FACS, MTT, SRB, apoptosis and necrosis assays)
actually, it should be determined what the target of the drug is
O i see,
That's sounds much more complicated. I thought there was a way to determine IC50 from MTT. Maybe the halfway point between the absorbance of positive control (cells with no drug treatment) and negative control (absorbance with media alone)...and the absorbance at half point should give me the concentration of drug at IC50?
yes, technically right but the IC50 determined with MTT only says that at a certain conc of your drug under defined conditions 50 % of your cell are living; there will be the question how to interpret this finding, what are the reasons for it, and therefore, you need additional methods as I suggested;
for a start MTT assay it is okay;
Right,
I agree, I just wanted to make sure with the MTT assay first at what concentration are the drugs living at 50% and how to extrapolate that concentration with the graphs. Thanks for your suggestions!