I have transduce HEK293T cell lines and am attempting to characterize some cellular and mitochondrial phenotypes after KD of my gene of interest. Seahorse is one of my main proposed assays but I can't seem to get acceptable results in terms of my variation and error bar sizes. They are literally > 100 units +/- for both ECAR and OCR. I have tried a range of about 4 cell densities from 1X10^4 - 2X10^5 and found a good one around 4X10^4 for 50-90% confluence 12 hours (O/N) before starting. I have tried 3-6 replicates (wells) per sample. I coat the plates with Poly-D lysine before since HEK are so loosely adherent and I reduced protocol mixing to a minimum of 1 so the plate is not shaken so much. That seemed to help a lot but still not enough. I always check the cells before and after and I normalize by cell number using CyQuant which should fix a lot of the problem too. Also, I just purchased CellTak since so many scientists have sworn by it but haven't had the chance to test with this. The only other thing I can think of to troubleshoot this is to try another cell line known to work well as a positive control. I wonder, do others experience such insane variation too and just remove outliers to produce nice data or is it just me? If I did want to go and remove outliers, is it possible within the wave software or would I need to go through and manually analyze the raw data?
Thank you for any advice in advance,
-A desperate PhD student
Contacted Agilent to help troubleshoot my variation and they were very helpful, specifically looked into my raw data file and was able to tell me that the machine from my core facility hasn't had a check up in two years (it's already pretty old but can still generate good data) and that my media is acidic so maybe the media isn't being pH checked or buffered properly. Also, I will do a complete titration of my cells to make sure I am using the optimal density.
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