It sounds like you are on the right track, as for tracking specifically mitochondrial derived ROS your options are limited. As you mention, there are some roGFP and HyPER proteins that can be targeted to the mitochondrial, and are truly the best bet for steady-state mitochondrial ROS measurement. Due to the fact that you have primary hepatocytes, and most likely do not have these constructs in an adeno or lentivirus (works well on hepatocytes), you will be limited to ROS sensitive dyes.

MitoSOX red is a good dye for mitochondrial superoxide measurement, but you state that mtDNA is less in your KO hepatocytes (suggesting fewer mitochondria). A way around this would be to do a MitoTracker Green test along with your MitoSOX. MitoTracker green is insensitive to ROS, and as such will give you a representative level of mitochondria mass in your cells (and it should be lower in your KO cells as you have demonstrated lower mtDNA, and will be a great piece of supporting data as well). By doing this, you can divide the MitoSOX MFI by the MitoTracker Green MFI to get a representative ROS per mitochondrion in your cells.

This method is very similar to what people do when performing mitochondrial membrane potential with the JC-1 dye. The JC-1 dye is sensitive to membrane potential, but you also run a second MitoTracker Green dye that is insensitive to any type of oxidation or membrane potential change. In this manner, you can observe a mitochondrial membrane potential per mitochondrion in your cells.

This will probably be your best bet at this point with your cells. Good luck!

~Adam

It sounds like you are on the right track, as for tracking specifically mitochondrial derived ROS your options are limited. As you mention, there are some roGFP and HyPER proteins that can be targeted to the mitochondrial, and are truly the best bet for steady-state mitochondrial ROS measurement. Due to the fact that you have primary hepatocytes, and most likely do not have these constructs in an adeno or lentivirus (works well on hepatocytes), you will be limited to ROS sensitive dyes.

MitoSOX red is a good dye for mitochondrial superoxide measurement, but you state that mtDNA is less in your KO hepatocytes (suggesting fewer mitochondria). A way around this would be to do a MitoTracker Green test along with your MitoSOX. MitoTracker green is insensitive to ROS, and as such will give you a representative level of mitochondria mass in your cells (and it should be lower in your KO cells as you have demonstrated lower mtDNA, and will be a great piece of supporting data as well). By doing this, you can divide the MitoSOX MFI by the MitoTracker Green MFI to get a representative ROS per mitochondrion in your cells.

This method is very similar to what people do when performing mitochondrial membrane potential with the JC-1 dye. The JC-1 dye is sensitive to membrane potential, but you also run a second MitoTracker Green dye that is insensitive to any type of oxidation or membrane potential change. In this manner, you can observe a mitochondrial membrane potential per mitochondrion in your cells.

This will probably be your best bet at this point with your cells. Good luck!

~Adam

Thanks Adam, for such a detailed reply.

I have already tried Mitotracker Green but the number and volume of the mitochondria in our mice are increased while mtDNA is decreased thus in this case taking the ratio of MitoSox/MitoTracker is not a good option I guess as it will further ameliorate ROS observed and thereby compromising the reliability of the data/experiment.

Thanks again for your inputs.

Best regards,

Abhijit

I still think it is a good option. Even if it ameliorates the ROS signal when you normalize to mitochondrial number/volume, that is important data! Contrary to popular belief, ROS are not involved in EVERY disease or model. If you show that you have a change in mtDNA and mitochondrial number/volume, but not a change in mitochondrial ROS per mitochondrion that is VERY interesting and important. Follow the data, not your hypotheses my friend. Good luck.

~Adam

Ya I agree with you completely Adam, but what I am trying to say is that the mtDNA is already low in our KO (we have the data for this). The principle of MitoSox is that it gets oxidized in mitochondria, binds to mtDNA and gives fluorescence. So my concern here is that as mtDNA is already low is ROS data given by MitoSox is reliable? Because this could be a limiting step. It could be that there is more oxidized dye but you do not have enough mtDNA to bind with and you see less ROS or it could also be the other way round with actually less ROS and you still have some vacant mtDNA (we have seen less ROS in KO cells) and that is why we see less ROS, but there is no confirmation for this.

I hope you understand what I am trying to say! If i have a solution to explain this problem, it could be a great addition to the work done.

Abhijit

Measure the superoxide with MitoSox, peroxides with H2DCFA and hydroxyl radical with HPR.

You can also measure mitochondrial DNA damage using Fpg-ddTUNEL

(see my refs).

Always do a positive control by adding paraquat. This allows you to know the dynamic range of your system.

Yes, I understand your concern now. I am assuming your quantified mtDNA by some sort of PCR reaction and normalized it to genomic DNA, correct? You could potentially use this as your normalization method if you are concerned that the decreased mtDNA would mean decreased MitoSOX oxidation. This method may not be perfect though as the decrease observed in mtDNA may not be completely 1:1 with a potential decrease in MitoSOX oxidation. It could be worth a shot, but may also difficult to interpret.

You may want to look into obtaining the roGFP or HyPER constructs in a viral form, as I know they exist. Another option is to contact Chris Chang's group for some MitoPY1 (http://www.nature.com/nprot/journal/v8/n6/abs/nprot.2013.064.html). This is a mitochondrial localized boronate compound that is specific to H2O2 (if you are interested in measuring it). One caveat to these probes is that they also measure peroxynitrite (ONOO-) very well, so usually people use them in the presence/absence of uric acid, which is a ONOO- scavenger (meaning if the fluorescence does not change +/- uric acid, then its most likely H2O2... if it does change, then its most likely ONOO-).

Thats all I can think of for now, good luck.

~Adam

Hi Adam, yes you got it correctly. Thank you so much for all the inputs, I will look in to all the possibilities you suggested.

Best regards,

Abhijit

Thank you Martyn, I will look in to the things you suggested.

Best regards,

Abhijit

Hi Abhijit, you can try to stain pico green (PG) for staining double-stranded DNA both in the mitochondria and nuclei in the presence of mitoSOX red. I have used H2DCFDA, dihydrorhodamine, mitoSOX and JC-1 to pin point how mitoROS affects mitochondrial biogenesis or degeneration to induce apoptosis. This is based on the qualitative assessment when you need a quantitative measurement, you can use fluorometer to read both green and red directly on the plate. Alternatively you can measure them after cell lysis by ultrasonication. Thanks.

Dear Syed,

Thank you so much for your inputs. The data that you are talking about, i mean mitoROS and mito genesis; is it published already? If yes, can you please give me the link or the PMID for the same? That would be a great help.

Thanks in advance,

Best regards,

Abhijit

Thanks Abhijit, we have been writing up the paper but you can get some idea from my recent published paper in Journal of Autoimmunity 2013 (Morshed SA et al.). In the current paper, I have added how mitoROS can induce ER stress during GPCR signaling. The methodology would be similar to what I described in that journal. If you need technical help let me know.

Deae Abhijit I think you have very good suggestions; however, I thinl Rhodamine 123 could be a good option for your experiments. The use of mitoROS is also a good option, good luck

Thanks Syed, I will look it up.

Thanks a lot Juan, for your inputs as well!

Abhijit

Dear Abhijit

Rhodamin 123 can be used in MMP, For ROS you can use DCFH-DA (10 uM) dye 30 mins prior to treatment.

Hi everybody,

Abhijit, did you try to use the histochemical method to monitor ROS in tissues?

Method is described and pictures are shown in D'Errico I. et al. PNAS April 2011.

Lorena

I think you would not need a better answer than that given by Dr. Adam Case.

Hi Lorena,

Thanks for your inputs! I will look it up.

Yes indeed Jai, I had a good discussion with Adam in this post.

Thank you everybody for all your inputs!

Abhijit

Hi Abhijit. Boveris A et al are well known authors in this field and I think that you must consult them in PUBMED.

The mitochondrial membrane Potential assays using Rhodamin 123 is certainly interesting. However, Rhodamin-123 requires carrier-mediated influx for its activity. Is it possible that the transporter activity is hampered by the used set-up?