Hi Suleyman,

Although it is not my field of expertise, I have done some related experiments. I want to share our protocol which might help you:

1) As we do in every Flow Cytometer (FC) staining, we prepare an unstained control and a positive control for every single experiment. The unstained control is for detecting the false peak - a gate should be placed at the right end of the signal (this might be differently visualized in different FCs). And by this way you may have an idea about whether your staining worked or not (or how efficient the staining was).

2) MitoTracker (MT) staining incubation time should be around 30 to 45 min. An optimization step for dose detection should be done before actual experiments by testing 20 to 200 nM MitoTracker. We choose the right concentration by looking at the staining efficiency of each dose. Roughly, all of the cells should be separated from the false peak (just like in your 100 nM staining).

3) The positive control is CCCP or FCCP. Briefly, we use CCCP treatment (4 hours, 10 uM) as a positive control to demonstrate collapsed ΔΨm, which is indicated by a clear decrease in MitoTracker staining. If you see a decrease of mitochondrial mass after CCCP treatment, then your setup is well established. There are many papers about using CCCP as a positive control, please have a look at NCBI. Here is one of them: https://www.nature.com/articles/srep22997

4) At the end, by looking at the mean values (obtained from the FC) of each peaks you can do statistical analysis: Untreated vs treated.

If you have any additional question, do not hesitate to contact me.

Good luck and all the best for your future experiments,

Ulvi

Hey suleyman,

I might be wrong but here i have some suggestions

Flow cytometry is a game of different color matching and you have to make sure that there is no an overlap in your color design of you staining dyes and surface markers and the following link might help you to identify what is what.

https://www.thermofisher.com/au/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html

your dye concentration depends on the number of cells that you are staining and optimizing with different conc. is recommended and different time point as well. good luck

Hello Suleyman,

maybe the discussion in the forum 'Protocol online' can help you with your question:

http://www.protocol-online.org/biology-forums-2/posts/10121.html

With best regards,

Christian

Dear Suleyman, Yes I see your problem I think you need turn up the voltage so the control mitochondrial fluorescence is higher up the scale as the concentration used is fine (just below 104 would be good). Unfortunately you did not show a profile of your drug treated cells these as you say would be lower if the drug has caused mitochondrial inner membrane potential to be depolarized (not as low as unstained control though) and higher than the control if you have hyperpolarization). I have an example on my website that might be helpful http://www.icms.qmul.ac.uk/flowcytometry/uses/apoptosis/diagrams/mmp.gif

http://www.icms.qmul.ac.uk/flowcytometry/uses/necroptosis/necrostatin.gif

Regards

Gary

Hi Suleyman,

Although it is not my field of expertise, I have done some related experiments. I want to share our protocol which might help you:

1) As we do in every Flow Cytometer (FC) staining, we prepare an unstained control and a positive control for every single experiment. The unstained control is for detecting the false peak - a gate should be placed at the right end of the signal (this might be differently visualized in different FCs). And by this way you may have an idea about whether your staining worked or not (or how efficient the staining was).

2) MitoTracker (MT) staining incubation time should be around 30 to 45 min. An optimization step for dose detection should be done before actual experiments by testing 20 to 200 nM MitoTracker. We choose the right concentration by looking at the staining efficiency of each dose. Roughly, all of the cells should be separated from the false peak (just like in your 100 nM staining).

3) The positive control is CCCP or FCCP. Briefly, we use CCCP treatment (4 hours, 10 uM) as a positive control to demonstrate collapsed ΔΨm, which is indicated by a clear decrease in MitoTracker staining. If you see a decrease of mitochondrial mass after CCCP treatment, then your setup is well established. There are many papers about using CCCP as a positive control, please have a look at NCBI. Here is one of them: https://www.nature.com/articles/srep22997

4) At the end, by looking at the mean values (obtained from the FC) of each peaks you can do statistical analysis: Untreated vs treated.

If you have any additional question, do not hesitate to contact me.

Good luck and all the best for your future experiments,

Ulvi

Hi

Suggestions:

1- check the flow channel (filter). I saw you have used

PerCP-Cy™5.5 (Ex-Max 482 nm/Em-Max 676 nm) and MitoTracker® Red CMXRos (M7512) EX/Em are ( ex- 579nm/ em- 599nm).

2- You may need to use MitoTracker® Green FM M7514 † 490 516 to evaluate Mitochondrial mass. Your treatment may affect the number of Mitochondria in a cell ( Mitochondrial mass).

3- As suggested above, use FCCP as a positive control. I have used 20nM FCCP for 10 min in a prior to staining which takes 30min. Using high concentration of FCCP cause problem to the cells.

Good luck

Unexpected results when using MitoTracker Green FM and CCCP treatment in cytometry. How to explain?

Unexpected results when using MitoTracker Green FM and CCCP treatment in cytometry. How to explain?

We want to evaluate mitochondrial mass. Cells were stained with MitoTracker Green FM (Cat. M7514), whose fluorescence monitors total mitochondrial mass and in particular, we want to study mitochondrial mass at basal conditions and after the treatment with CCCP. Unexpectedly, we found that cells treated with CCCP at different concentrations, had a higher MTG signal.

We have repeated the experiments several times and we always get an increase in the MTG signal.

How can I explain such results?

I would be very grateful if someone could help to solve this question

Thanks in advance

Patricia García-Sanz generally You would get decrease intensity of the fluorescent. What is the name of the cell line? Do you count the cells before the stain? Equal number of the cells are important for the staining in my experience.

It almost seems that the cells respond with mitochondrial biogenesis after the CCCP treatment. I agree with Suleyman, first the experimental parameters need to be checked and confirmed. But if reproducible and not being an artifact these data surely are interesting.

Regards,

Christian

Also Can you check LC3B bands on the western blot or mitochondrial protein like Tom20 whether it will reduce or not. I strongly advice you to check dead and live on the CCCP treatment because it can cause apoptosis as well. Maybe you can try antimycin and oligomycin mix

Thank you very much for all your help. Answering your questions:

Our cells are human skin fibroblasts.

We counted all the cells the day before cytometry assay with a biorad cell counter specifically bought for that. We also did a cytometry test with IP to detect dead cells.

In the image we show you an article where fibroblasts are used. we contacted with the authors and they simply told us that they preloaded with the probe. We also tested it and we obtained the same results.

We previously did tests to determine the appropriate probe dose for our two cytometers:

1) CytoFLEX up to 3 lasers & 13 fluorescent detectors (488 nm, 638 nm, 405 nm): 0.5, 1, 2.5, 5, 10 and 100nM = 10nM

2) 10, 25, 50, 100nM using a FACS Aria I flow cytometer = 50nM

We also carried out a western blot of LC3 and Tom at the doses of 25uM of CCCP during 6 hours and you can see the results in the other image.

We do not know what is happening; the next step is to perform a similar assay using other types of cells. If you could give us some advice we would be very grateful

Patricia,

indeed very interesting result I must admit. Tom20 is also increased with CCCP treatment interestingly. I just suggest couple things but I dont know are they available or not.

1- MTT and LDH test for CCCP conc and time curve. (I suspect that cells try to grow)

2- MitoSox dye from Thermo. In my experience, CCCP increases mitosox dramatically like 10 fold.

3- I am also suspected that cells are not going to mitophagy at all or mitochondria biogenesis are higher than mitophagy rate. Seahorse respiration rate can be tested but this is quite expensive I know.

4- If you can transfect this cell, you may use MT-Keima (https://www.ncbi.nlm.nih.gov/pubmed/28093935) There are a lot of different options to control biogenesis and breakdown of mitochondria.

5- Mitochondria DNA copy number test.

6- Best way to use electron microscope to see mitochondria in the autophagasomes.

Some of the techniques that I mentioned quite expensive. But I hope you can try some of them. But I just recommend another mitophagy induction agent like mix of antimycin and oligomycin. In my system, I used 2.5uM of Oligomycin and 200nM Antimycin). I suggest you to look into Richard Youle's articles.

I hope this could help.

Best