My stock solution of Rotenone (Sigma) is 10mM in which 0.004g of Rot is diluted by 1mL of DMSO. I have done cck-8 assay on SH-SY5Y (2 x 10^4) with Rot treatments. Several articles show that 1uM of Rot treatment is toxic enough to kill the half of the whole cell population but my treatments up to 20uM barely kill them. I need a solution to this.
Thank you.
Hello Jong Seung Min
Rotenone, at nanomolar concentrations, induces apoptosis in SH-SY5Y cells. Do you use FBS in the culture media when you treat the cells with rotenone?
Rotenone is lipophilic and may bind to proteins present in the serum. Therefore, before rotenone treatment, the cells should be in media containing lower concentration of serum (for instance, 0.5% FBS) in order to prevent excessive retention of rotenone in the serum. Having low serum concentration in the culture media would not noticeably increase basal apoptosis in the cells in the absence of rotenone for at least up to 48 hours.
Best.
Due to the Warburg effect cancer cell lines typically rely on aerobic glycolysis for their ATP production. Since rotenone is a mitochondrial poison and the mitochondrion is relatively unimportant as an ATP source in these cell lines, a lower sensitivity to rotenone would be expected.
Hi Jong Seung Min
Rotenone is toxic to SH-SY5Y cells even at nano molar concentrations. If it does not work at 10uM too for you, then most probably it is degraded. Rotenone is light sensitive and multiple freeze thaws can get it denatured and form aggregates. So it is important to minimize the number of freeze thaws and prepare small stocks of the drug.
Hope it helps.
Thanks,
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