Dose Response Troubles - (Jan/18/2012 )
Hello All,
I have been performing dose response curves for certain chemotherapeutic that takes 3days to work on the cells. Earlier on a few months ago I was sucessful at obtaining nice curves with fairly low IC50 values. In the recent experiments I am not able to replicate my earlier results. My overall strategy is as follows, I have a stock solution of drug solubilized with DMSO at a stock concentration of 50mM. For cell culture, since DMSO is toxi, I vortex the solution and take 20uL of the stock and bring to volume with a 10mL volumetric flask using growth media which effectively is a X500 dilution. So the concentration would then be 100uM as the starting stock, I then do a series of 2X dilutions to get 50uM, 25uM, 12.5uM, 6.25uM, 3.125uM.
One thing which I have recently thought about which I am unsure of is the filtration step. Usually after the dilution into cell culture media I filter with a hydrophilic 0.2uM PVDF millipore filter using syringe into falcon tube. I am wondering if this is somehow trapping my drug and making that more dilute.
Earlier on I was obtaining IC50 values of ~5uM, recently that has shifted to around 50uM and something is really wrong with the results, as even the 100uM concentration has only around .50 response.
I am plating about 5,000 cells per well as 100uL in quadruplicate. Following day adding the drug solution, with incubation for 3 days, whereby I add MTT and check the results.
Any idea as to what is going on?
I already made a fresh solution and performed the experiment with similar results..... Could the filtration be an issue? Maybe the cells have been passed too long and have morphed into different state?
Any advice, tips or tricks ?
Thanks
shamash119 on Thu Jan 19 02:27:09 2012 said:
Hello All,
I have been performing dose response curves for certain chemotherapeutic that takes 3days to work on the cells. Earlier on a few months ago I was sucessful at obtaining nice curves with fairly low IC50 values. In the recent experiments I am not able to replicate my earlier results. My overall strategy is as follows, I have a stock solution of drug solubilized with DMSO at a stock concentration of 50mM. For cell culture, since DMSO is toxi, I vortex the solution and take 20uL of the stock and bring to volume with a 10mL volumetric flask using growth media which effectively is a X500 dilution. So the concentration would then be 100uM as the starting stock, I then do a series of 2X dilutions to get 50uM, 25uM, 12.5uM, 6.25uM, 3.125uM.
One thing which I have recently thought about which I am unsure of is the filtration step. Usually after the dilution into cell culture media I filter with a hydrophilic 0.2uM PVDF millipore filter using syringe into falcon tube. I am wondering if this is somehow trapping my drug and making that more dilute.
Earlier on I was obtaining IC50 values of ~5uM, recently that has shifted to around 50uM and something is really wrong with the results, as even the 100uM concentration has only around .50 response.
I am plating about 5,000 cells per well as 100uL in quadruplicate. Following day adding the drug solution, with incubation for 3 days, whereby I add MTT and check the results.
Any idea as to what is going on?
I already made a fresh solution and performed the experiment with similar results..... Could the filtration be an issue? Maybe the cells have been passed too long and have morphed into different state?
Any advice, tips or tricks ?
Thanks
Dear Shamash119,
Is your drug stable?
Have you stored it correctly?
Have you used the same type of filter? ...cellulose acetate, polycarbonate, PTFE, PVDF. Each type MAY collect/adsorb the drug differentially.
Have you used the same passage number of the cell line.......it is very important to check the stability of the cell/cell line.
Have you checked thoroughly the environment that the cells grow in .....for example checking CO2 concentration of the inside of the CO2 incubator.
Have you checked the pH of your cell growth medium......a 500ml bottle will gradually become alkaline....due to the fact that the atmospheric CO2 concentration is 0.03% and the media requires 5-10% CO2 concentration.
Are you sure that you have diluted the drug correctly?
Have you used the same Gilson pipette to do the dilutions?
There are some things that I can think of quickly, there are probably many more.
Hope this is useful
Kindest regards
Uncle Rhombus
Agree totally with uncle Rhombus,
Just one more thing: the drug, Check if the drug was purchased recently and if its from the same vendor. If its been around for a long time, order some more and try it.
Good luck !!!