Different drugs and different conditions have been tried on two cancerous cell lines named A549(epithelial) and U2OS(osteosarcoma). However, when using Rotenone initially diluted in DMSO but then placed onto full DMEM media to form a final concentration of 1uM,the resulting solution is viscous and not fully dissolved making colorimetric assay results unreliable. Any solutions to this? It is a well established drug used in classical studies into mitochondrial function so hoping someone will know.
According to Tocris, rotenone is soluble at 100mM in DMSO, so it's a bit surprising a 100,000X diluted solution of this (i.e. 1uM) would be viscous. I've worked with the structurally similar compounds alpha-toxicarol and degeulin , which were effective at inducing AMPK phosphorylation and inhibiting mTOR phosphorylation at 2.5uM as observed by western blot. I used these for high content screening experiments and even at 50uM concentration I saw no interference with DAPI or Alexa647 fluorescence, however cells were washed extensively with PBS prior to imaging. Maybe this is specific to your colorimetric assay? Maybe you can wash the cells prior to performing the assay?
According to Tocris, rotenone is soluble at 100mM in DMSO, so it's a bit surprising a 100,000X diluted solution of this (i.e. 1uM) would be viscous. I've worked with the structurally similar compounds alpha-toxicarol and degeulin , which were effective at inducing AMPK phosphorylation and inhibiting mTOR phosphorylation at 2.5uM as observed by western blot. I used these for high content screening experiments and even at 50uM concentration I saw no interference with DAPI or Alexa647 fluorescence, however cells were washed extensively with PBS prior to imaging. Maybe this is specific to your colorimetric assay? Maybe you can wash the cells prior to performing the assay?
Thanks . It may be because of the cell volume (100ul total volume,containing 1uM Rotenone).
Hi Abel,
I agree with Mark that it is a bit surprising that you get this effect at 1uM rotenone. I routinely make rotenone stocks at 1mM (soluliblized in DMSO), then treat cells in a range from 10nM to 2uM diluted in DMEM. These experiments are performed in 96- and 384-well plates, so volumes of 30ul to 100ul. Have you tried different vendors or different lots of the stock rotenone?
try solubilising with ethanol.. advantage ethanol has lower toxicity as well as non-viscous..
I had the same problem I have prepared stock 100 mM in DMSO but when I wanted to resuspend it to 250 uM in DMEM I couldn't obtain clear solution, I will try ethanol however the supplier (Sigma) recommend DMSO or Chloroform
Anna Tejchman "I had the same problem I have prepared stock 100 mM in DMSO but when I wanted to resuspend it to 250 uM in DMEM I couldn't obtain clear solution, "
The stated water solubility of rotenone is 0.0129 mg/ml = 3.27 uM.
Therefore 250 uM will indeed give a suspension which should dissolve upon further diluting this intermediate mixture below 3 uM.
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