Why don't you try other cellular stressors such as MPP+, dopamine, H2O2, Fe2+, proteasome inhibitors? Alternatively, you may use other neuronal cell lines (e.g. SH-SY5Y) which is more susceptable to rotenone.

Dear Professor Hashemi. I dissolved the rotenone in DMSO

Increase the micromoles of rotenone to 12, u can find cell mortality

Undifferentiated PC12 cells are not really good models of neurons because they still undergo mitosis. You could differentiate them for a few weeks until they become post-mitotic, then try your rotenone stress again. This is true also for SH-SY5Y and other "neuronal" cell lines. They only become "neuronal" when they are differentiated.

Also, be careful with rotenone. At high concentrations, rotenone begins to disassemble the actin cytoskeleton. So of course it will cause toxicity, but not because of Complex I inhibition. A better model of rotenone toxicity is very low concentrations for relatively long times. The IC50 of rotenone is 50nM (nanomoles per liter), so if you're using 12 micromoles (in what volume?) you are probably using 1000 times too much.

You should read some papers by Tim Greenamyre. He's done a lot of work with rotenone models, both in cell culture and in animals, and his work is very reliable.

Here's another paper by Dr. Greenamyre, to help get you started.

The MTT viability assay relies on metabolism of the MTT reagent in the mitochondria. Thus, it measures mitochondrial metabolism -- which is often a good indicator of cell viability, but the two are not the same. If you want to measure viability then you need to use another indicator, like LDH activity, to quantify cell death. Look at these two measures together (metabolism of living cells by MTT -- but doesn't tell you about the number of living cells -- plus number of dead cells from the LDH assay). Then you will be able to draw conclusions about the health and viability of your cells.

In fact, undifferentiated PC12 cells are not a good model for PD. You can differentiate with NGF to dopaminergic neurons. These neurons are now very sensitive to rotenone. With 500 nM rotenone, and after 24 h of incubation, you inhibit almost 90% of the complex I activity, yielding an impairment of mitochondrial function including increased oxidative stress, reduced ATP production, opening of the MPT, etc. In thse conditions, almost 50% of the cells die.

Differentiating PC12 decreases or stops the ability of PC12 cells to produce dopamine. Human SH-SY5Y cells also do not produce dopamine although they are TH positive. Therefore differentiated cells of either line do not truly represent dopaminergic neurons.

@ glenn mcConkey. Do you have the paper citation that states SH-SY5Y cell do not produce dopamine? I would be interested in reading it.. Or any other paper delineating the dopamine metabolism within sh-sy5y. thank you.

SK-N-MC cells produce dopamine and are available from ATCC. Not sure about differentiating them, though.