MitoTracker Red CMXRos has very poor absorption at 488 nm. It is a particularly
good imaging
stain because it localizes in the relatively small mitochondria but may be less
suitable in
flow cytometry, although it has been used to detect apoptotic populations in mostly
single-color experiments. It is optically transparent with the 633 nm laser, however,
so if
you get enough signal excited at 488 nm you could likely combine it with APC conjugates
etc.
Absorption and fluorescence emission spectra of MitoTracker Red CMXRos (M-7512)
in methanol.
[Spectra: 11KB]
Cytometry 1997 Apr 1;27(4):358-64
Detection of apoptosis in live cells by MitoTracker red CMXRos and
SYTO dye flow cytometry.
Poot M, Gibson LL, Singer VL.
Molecular Probes, Inc., Department of Biosciences, Eugene,
OR 97402,
USA.
We characterized the ability of six SYTO nucleic acid stains and a
mitochondrial stain to resolve by flow cytometry
camptothecin-induced apoptotic and non-apoptotic cells. Staining
live
human lymphoid B-cells showed such resolution with SYTO 11,
12, 13, 14, and 16 dyes. H9, HL-60, and Jurkat cells did not show
resolution with the SYTO 12 dye, but did with the others. SYTO
15 dye did not show resolution with any cell type. RNase
A treatment
of fixed lymphoid B-cells strongly reduced fluorescence after
staining with SYTO 12 dye; the other SYTO dyes showed little or no
RNase A sensitivity. Reduced SYTO 12 fluorescence may
reflect RNA breakdown during apoptosis, while decreased
fluorescence
of the other SYTO dyes in apoptotic cells may be due to
chromosomal alterations during apoptosis. In all cell types
tested,
clear resolution between apoptotic and non-apoptotic cells was
observed with the MitoTracker Red dye CMXRos. In double-staining
experiments, cells exhibiting reduced SYTO 11 fluorescence
were the same as those showing decreased CMXRos fluorescence. We
conclude that changes in nucleic acid stability or conformation
may vary during apoptosis from one cell type to another, but
mitochondrial demise may be common to all apoptotic pathways.
MitoTracker® Deep Red 633, is a totally new probe that may be useful with the He-Ne
laser.
It has maximal absorption at 640 nm and emission at 662 nm in methanol. We have used
it in
imaging but not flow and it localizes well in mitochondria. There are no papers
published on
it yet, however.
Annie Bang wrote:
> Hi,
>
> Has anyone had flow experiences with Molecular Probes' product MitoTracker Red
(CMXRos),
> in particular with 3-colour staining?
>
> We are using a FACSCalibur with a 488 nm laser and find that the fluorescence of
> CMXRos alone is detected in FL2, FL3 and FL4. We can decrease the FL2 signal to a
> negative level using FL2 - 99.9% FL3 compensation while retaining the signal in FL3
> and FL4. However, we cannot decrease the signal in FL3 using FL3 - 99.9% FL2 (or FL4)
> compensation.
>
> I am having a hard time conceptualizing this compensation model. Any ideas?
>
> Thanks so much.
>
> Annie Bang
> Platelet Research Laboratory
> St. Michael's Hospital
> Room 2-001
> Cardinal Carter Wing
> 30 Bond Street
> Toronto, Ontario
> M5B 1W8
> Canada
>
> 416-360-4000 ext 2192
> 416-864-3060 fax
> banga@smh.toronto.on.ca
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:29 EST