Can I scale up at transfection, after determining the drug concentration via kil - (Mar/15/2013 )
I will perform stable tranfection in 6-well plates and my selective drug will be Geneticin. Can I perform the kill curve at 24-well or 48-well plates, and then scale up to 6-wells for transfection? My supervisor is suspicious about scaling up, and thinks the activity at determined concentration may be affected by number of cells, volume of the media, surface and so on. Has anyone ever tried this? Thank you.
Your PI is right. Your added drug concentration needs to consider the total volume of your media to obtain a final drug concentration. Surface should have no affect. Most plates are washed to provide an etched surface for cells to adhere to. Do you have any previous information or can you find any information online about a relative kill concentration?
I would just innoculate a six well plate and do 50, 100, 200, 400, 800, etc mg/mL of your G418. From here, you can find the lowest drug concentration that kills cells, but allows continued proliferation. If need be you can fraction this found kill concentration and modify it further. i.e, if you found that 200-40 is the lowest drug dose that kills, but maintains normal growth. From here maybe try 200, 225, 250, etc.
The most important thing to remember is that the higher the drug concentration (although it will rapidly kill cells and provide the results you acquire) is not the absolute best.
Important to run your vector only control side-by-side to assure efficient uptake of your vector (make sure your insert is not being fussy).
Edit: Oops. It is always good to reference the ATCC website to obtain info about your cell line.
Many thanks for all the information you shared, helped a lot.