If you are doing cell imaging, a preloading or pretreatment step (1 h) is recommended. 

mitoTEMPO carries a triphenylphosphonium moiety for mitochondria-targeting. It takes a while for the compound to accumulate to effective concentration in mitochondria (more precisely on the inner membrane). 

http://www.enzolifesciences.com/ALX-430-150/mito-tempo/

Typically for selective use, 2-10 uM should be enough, unless your superoxide inducer is very strong. 

Since TEMPO / 4-hydroxy-TEMPO is a scaveger, it should be added before you do the superoxide induction, and during induction, the scavenger should also be there (not washed off). Scavenging is competitive in nature, and so a wash step would be counterproductive. 

A positive control would be antimycin A, 500 nM to 1 uM. mito-TEMPO may not be able to deal with higher doses of strong stimulants. 

There are papers using high doses of mito-TEMPO as high as 100 uM or more. That's not recommended, because it introduced nonselectivity. I would say max 20 uM. 

See for an example: 

http://www.ncbi.nlm.nih.gov/pubmed/18662543

>Secondly, I found that in some references MitoTEMPO is added along with the stress inducer and MitoSox at the final hour of the stress inducer treatment NOT after completion of the treatment. 

Exactly, this is a wrong way of using a scavenger. 

Good luck!

If you are doing cell imaging, a preloading or pretreatment step (1 h) is recommended. 

mitoTEMPO carries a triphenylphosphonium moiety for mitochondria-targeting. It takes a while for the compound to accumulate to effective concentration in mitochondria (more precisely on the inner membrane). 

http://www.enzolifesciences.com/ALX-430-150/mito-tempo/

Typically for selective use, 2-10 uM should be enough, unless your superoxide inducer is very strong. 

Since TEMPO / 4-hydroxy-TEMPO is a scaveger, it should be added before you do the superoxide induction, and during induction, the scavenger should also be there (not washed off). Scavenging is competitive in nature, and so a wash step would be counterproductive. 

A positive control would be antimycin A, 500 nM to 1 uM. mito-TEMPO may not be able to deal with higher doses of strong stimulants. 

There are papers using high doses of mito-TEMPO as high as 100 uM or more. That's not recommended, because it introduced nonselectivity. I would say max 20 uM. 

See for an example: 

http://www.ncbi.nlm.nih.gov/pubmed/18662543

>Secondly, I found that in some references MitoTEMPO is added along with the stress inducer and MitoSox at the final hour of the stress inducer treatment NOT after completion of the treatment. 

Exactly, this is a wrong way of using a scavenger. 

Good luck!

Hi Nai-Kei Wong,

Thanks for your anwser ! So in short, I should put mito- TEMPO along with my free fatty acid treatment. Do you suggest to add even before giving FFA treatment ??

hello, what I would suggest is, add mito-Tempo first (1h; or at least 30 min) at 2x the final conc. (e.g. 20 uM preloading; 10 uM final), in 1/2 of the final volume (say 96-well) -- say, 50uL per well in 96-well plate. 

Then, for the real treatment, add your stimulants (2x the final concentration), but also in 1/2 of the final volume. 

Meaning that you add only once the scavenger. 

And at the end of the treatment [say 1h or longer] (it becomes co-treatment with scavenger and stimulants), do a readout. 

This method is what I and my junior trainees have tried out. 

***

Another more wasteful method is:

preloading mitoTEMPO at working conc. (100uL).

After preloading (30-60min), suck out all the liquid from a well. 

Then add the stimulant (at working conc.) and mitoTEMPO (at working conc.) to a final volume of 100uL per well. 

This method is somewhat simpler, but certainly costs more of the scavenger. 

Thanks a ton !! One last question: what do you use to give treatment as in DMEM or buffer ? Right now I am using DMEM without phenol red and glucose for washing and treatment because my cells come out during the treatment. Please suggest something on this. Thanks again !

the drug treatment should normally be done in medium (at serum level appropriate for your study). In buffer, if the drug treatment duration is too long (>1h) it becomes a starvation model. So, by default, buffer is not a first choice. However, some probes require that the detection be done in buffer. Since all groups are subject to the same probe incubation procedure, any effects (e.g. within 30 min in buffer) can be justified by this normalized procedure. I think it's acceptable. Yes, phenol red free medium is recommended (phenol red gives a green max emission, it may even have a tailing effect in red region, though I didn't test that in imaging). 

After sufficiently long pre-incubation (preloading) mitoTEMPO should be retained sufficiently enough as an intervention group. If you really need to have a wash step before readout, you can wash with HBSS or PBS quickly, then do the readout (with equal volume [.e.g 100 uL] of buffer or medium [no phenol red] of choice. 

A good assay needs optimization and some patient trial and error... 

Good luck on exp. 

Yes.. Patient trial and error.. Will definitely try these things. Its a great help, thank you again Nai-Kei !

No problem. Hope your exp. goes well!

MitoTEMPO  is not phenol red sensitive, is it ??

According to a chemist labmate here, phenol red potentially can react with TEMPO (which is a stable nitroxide radical, and can react with weak reducants such as superoxide). 

Therefore, to play safe, it's better to buy a bottle of phenol red free medium / reagents for your treatment exp. cheers

So do you use DMEM without phenol red for treatment of TEMPO along with stress inducer ? 

I don't do overnight treatment often anymore. As I recall, I did my drug treatment normally in phenol red free medium. The dye-free medium is not much more costly than the one with the dye. Order it from Invitrogen/Gibco. 

For probe incubation, I use HBSS (Invitrogen), in place of PBS. 

Hi Nai-Kei,

I tried your method of 2x pre-loading but surprisingly I am getting almost equal or higher ROS production in TEMPO treated wells than untreated. I used phenol red free medium for the treatment (which is 18 hours long treatment with fatty acids). What might be wrong ??

Thanks,

Rashmi

Hello Rashmi, first thing to check is whether you have the right mito-TEMPO source. I got mine from Sigma. I once bought from Santa Cruz (same as Enzo) but I found that the mito-TEMPO from the latter is not very good (up to 10x less potent than expected). I used Sigma as a source in the end. 

Secondly, be sure that your positive control is not too strong. 

In my mito-superoxide imaging experiments, (not yet published), I used antimycin A as a positive control. Depending on the cell type, AA effects can range from moderate to extremely strong. So some prelim tests on dose dependency is needed. Usually 0.5 uM to 10 uM is an effective range. Once you know which dose give a substantial effect, plan for the scavenger group. I found that despite relative selectivity, you cannot use mito-TEMPO to block all mito-ROS. This is because: mito-TEMPO go to mitochondrial inner membrane based on membrane potential (MP). When MP is low, mito-TEMPO itself cannot accumulate in mitochondria efficiently (if at all). So make sure, for comparison, you don't use an overly strong positive control (I would use 0.2 or 0.5 uM AA for the mito-TEMPO group, for example).

In principle, scavenging reaction is competitive in nature. The scavenger must be present in excess amounts to remove the substrate (in this case, superoxide) fast enough, compared to ROS production rate. Therefore, a third troubleshooting way is to increase the mito-TEMPO dose, 2x to 4x the amount you are using. 

Lastly, if you are using MitoSOX for measurement, be sure you don't use the probe at > 2.5 uM. At 5 uM, the probe itself is considered toxic when incubated for > 15 min. 

Because we use different cell models and stimulants, the experience I had cannot be directly copied. Hope this helps. Best

Hi,

I have also problem with the effect of MitoTempo. I tried different concentrations (from nM to microM) of MitoTempo (using 1 hour pre-incubation) but it wasn't able to eliminate the mitochondrial ROS generated by Antimycin (0.1, 0.5, 5 microM for 6 hours) treatments. In these experiments I stained the cells with MitoSox Red before the treatments. After that I supposed that it would be better to stain the cells after the treatments, but in this case I didn't detect fluorescence signals.  Maybe the mitochondrial membrane potential was altered by Antimycin blocking the penetration of MitoSox into the mitochondria, or I don't know. Are you using MitoSox-filled cells or what is the right protocol? Please, help! Thanks!

hello Kitti, Yes, I agree on this point:

"Maybe the mitochondrial membrane potential was altered by Antimycin blocking the penetration of MitoSox into the mitochondria, or I don't know."

Overproduction of AA-induced superoxide (and derivative ROS) may subsequently dampen the MMP, which reduces the antioxidant buffer capacity of mito-TEMPO over time. 

In our recent paper, we showed that antimycin A at 5 uM rapdily induces superoxide to high levels in just 15 min or less. 

Being a scavenger that works competitively, the scavenging rate by mito-TEMPO may lag behind AA's induction rate.  

So what's the right protocol then ? It's better to give treatment and then give MitoTempo treatment ?? Because what's optimal is not clear.

Dear Nai-Kei, I also have problems with mitoTEMPO. I showed that the drugs of interest induced mitochondrial superoxide and I would like to check if the cytotoxicity is depending on the superoxide induction. I have mitoTEMPOL from Cayman chemical and I pere-treat cancer cells with it (10-50um for 2-24 h) and then co-treat with the drug and mitotempol (10um) for 24h. Then I check cell viability by MTT. I do not see any change in cell viability for all the compounds. I tried to do this experiment with 2h drug incubation time in comparison with antimycin 2.5um and rotenone 5 um, antymycin induced 15-20% cell death, but it was not affected by mitotempol. How can I troubleshoot my experiment?