Drug titration

I did this experiment to determine the minimum amount of hygromycin I could use to select transfectants. It's not a bad idea to do a drug kill curve every now and then anyway as the sensitivity of your parasites to a drug can change when FCS batches etc change. I used HygroGold for this - it seems to be higher purity and less batch-batch variation than the standard stuff. I have similar data somewhere for blastocidin. I do plan to do this for all of the drugs that we use at some point - it's quite useful information. The experiment was designed thusly: 1. A wild type (i.e. sensitive to hygromycin) and a drug resistant cell line were maintained in culture in exponential growth for a few days. For the wild type, I used a procyclic form 927 cell lines that we use routinely in the lab. For the hygromycin resistant, I chose a cell line that was expressing the drug resistance cassette using RNA polymerase II read through transcription and using the short aldolase 3' UTR. 2. Both cell lines were split down to 1x10^5 cells.ml-1 and incubated with the following concentrations of hygromycin B (ug/ml): 0, 1, 2, 5, 10, 15, 20, 25, 50, 75, 100, 200, 500 3. The cell density was measured after 48 hours and the growth factor was calculated 4. The growth factor was expressed as a factor of the 0 ug/ml hygromycin control 5. The data was plotted to calculate the EC50 of the wild type and hygromycin resistant cells Hygromycin drug titration Wild type hygromycin EC50: 10ug/ml Hygromycin resistant EC50: 40ug/ml I was quite suprised how how 'unresistant' the hygromycin resistant cells actually were - presumably if you use a T7 or polI promoter to drive the hygromycin resistance cassette you will get a more resistant cell line. Your mileage may vary - let me know if you have done a similar titration.

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