Re: [DIYbio] Re: How does MitoTracker Bind?

Maybe it's just on my end but that paper you linked doesn't load and
gives an error, or the paper the person you forwarded linked.

Here is the invitrogen info packet on the MitoTracker stains

http://probes.invitrogen.com/media/pis/mp07510.pdf

"For the MitoTracker® Green FM probes, use a slightly lower
concentration (20–200 nM). At higher
concentrations, these probes tend to stain other cellular structures. "

and from here, potentially useful
http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Probes-for-Organelles/Probes-for-Mitochondria.html#head2

"MitoTracker Green FM Dye

Mitochondria in cells stained with nanomolar concentrations of
MitoTracker Green FM dye (M7514, ) exhibit bright green,
fluorescein-like fluorescence (, , ). The MitoTracker Green FM probe
has the added advantage that it is essentially nonfluorescent in
aqueous solutions and only becomes fluorescent once it accumulates in
the lipid environment of mitochondria. Hence, background fluorescence
is negligible, enabling researchers to clearly visualize mitochondria
in live cells immediately following addition of the stain, without a
wash step.

Unlike MitoTracker Orange CMTMRos and MitoTracker Red CMXRos, the
MitoTracker Green FM probe appears to preferentially accumulate in
mitochondria regardless of mitochondrial membrane potential in certain
cell types, making it a possible tool for determining mitochondrial
mass. Furthermore, the MitoTracker Green FM dye is substantially more
photostable than the widely used rhodamine 123 fluorescent dye and
produces a brighter, more mitochondrion-selective signal at lower
concentrations. Because its emission maximum is blue-shifted
approximately 10 nm relative to the emission maximum of rhodamine 123,
the MitoTracker Green FM dye produces a fluorescent staining pattern
that should be better resolved from that of red-fluorescent probes in
double-labeling experiments. The mitochondrial proteins that are
selectively labeled by the MitoTracker Green FM reagent have been
separated by capillary electrophoresis."


The MitoGreen FM isn't retained after fixation, and seems to
necessitate using smaller concentrations to avoid non mitochondrial
specific lipid binding. It seems that the other MitoTrackers are
based on fluorescence of the oxidized state of the dye after it
couples with cysteine residues, or is oxidized in the
mitochondria...but this MitoGreen FM and the other "FM" trackers only
fluoresce when they accumulate in lipid deposits.

As the forwarded original poster suspected, there might be some
liposome/micelle formation going on which is picking up the stain and
not allowing any of it to reach the mitochondria.

Have they tried another mito tracker stain besides the Green FM? It
seems to be the most finicky tracker dye after reading the info
packet, and requires a very specific experimental protocol as listed
in invitrogens attached packet.

The Mitochondria has two membranes and a lot of internal surface area
so I can imagine that the largest, if not perhaps 2nd largest
concentration of lipids within the cell membrane lipid bilayer itself
would be the mitochondria or the endoplasmic reticulum. Just a guess
though

Just wrote all this thinking it was forwarded from a stranger outside
the list...so might have rambled on a bit more than needed after
realizing it seemed to be a question already posted by Nathan or
PhillyJ.

It'd be neat to see some pictures of the failed/successful stains though.

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