Re: Apoptosis detection with Annexin V and DiOC6 -Reply

Howard Shapiro (
Thu, 23 Jan 1997 01:31:14 -0500 (EST)

Gerhard Nebe-von-Caron recently wrote:

> If I remember right from Howards articles on MP, the
> lipophilic character of the carbocyanines results in poor
> cytoplasmic diffusion, hence cytoplasmic MP whilst Rhodamine
> goes for mitochondrial MP if the free dye is washed away.
> Could that difference in 'clearance' time explain the
> differences in delay between depolarization and annexin V
> between the groups? Howard also commented on effects of for
> example proteins in the medium... changing the staining
> kinetic. I don't work on apoptosis yet and don't know the
> staining protocols, but I wouldn't be to surprised to find
> out that the death of the mitochondria follows the same path
> as in bacteria and stands in the beginning of that pathway.
The key difference between "cytoplasmic" and "mitochondrial" MP measurement
with cationic dyes, including cyanines, is the wash step. This was
demonstrated to a small audience (Lan Bo Chen's lab and mine) in 1980, when
we swapped dyes and I showed you could measure "cytoplasmic" MP with R123,
using lower concentrations than he normally used to stain mitochondria and
eliminating the wash, while he was able to get the same mitochondrial
staining with cyanines as he had with R123 by washing the cells. This was
so despite the differences in lipophilicity between R123, which is
relatively water soluble, and the cyanines, which get progressively more
lipophilic as you go from DiYC1(m) to DiYC6(m). DiYC7(m)and longer chain
cyanines start to lock into the membrane bilayer and act more as tracking
dyes than as potential probes.

When you equilibrate cells with DiOC6(3), the lipophilicity contributes as
much or more to the concentration gradient as does the Nernstian response to
MP. In red cells, which have an MP below 60 mV, which would generate less
than a 10:1 ratio of interior and exterior dyes concentrations, Sims et al
noted that the actual concentration gradient was several thousand to one.
If you use 10-50 nM dye, the concentration in the cytoplasm would be at
least 10-50 uM. The additional gradient expected between cytoplasm and
mitochondria would be somewher between 10:1 and 100:1; at these
concentrations, most intramitochondrial dye would be quenched, and the
signal from cells in equilibrium would therefore primarily reflect
cytoplasmic MP. Dye efflux due to pumps may shift the equilibrium somewhat,
but, as long as cells are left in dye solution, they remain stained.

If you wash the cells after equilibration with cyanines, it would be
expected that the mitochondria would retain dye more easily than the
cytoplasm because of the additional MP gradient; however, the
intramitochondrial concentration evidently drops enough so that the
mitochondria become fluorescent. Once dye is removed from the solution,
however, active efflux pumps, if present, will clear it from both
mitochondrial and cytoplasmic compartments.

Rhodamine 123 is considerably less lipophilic than cyanines, and the
concentration gradient between cells and the exterior is probably closer to
Nernstian (Leslie Loew and coworkers have demonstrated Nernstian behavior
for related rhodamine dyes). In order to load cells with R123, one
generally incubates them with at least 10 uM dye; the dye in solution is
sufficiently bright that cells can't be identified under the microscope or
in the flow cytometer due to the background fluorescence. Once the cells
have been washed, dye retained in mitochondria is clearly visible. To
measure "cytoplasmic" MP with R123, it is necessary to use a lower dye
concentration (100 nM or so), and keep cells in equilibrium with dye.

At this point, you might wonder why one shouldn't just use oxonols instead
of cyanines or R123. Since oxonols are anionic, and are (or should be)
therefore excluded from cells with interior negative membrane potentials,
they should, in theory, provide a "purer" measurement of cytoplasmic MP.
Most of the fluorescence from cells equilibrated with oxonol dyes appears to
come from the cytoplasmic membrane. However, there is evidence, not yet
published, that leads me to suspect that oxonols respond to changes in
membrane phospholipid structure as well as to changes in membrane potential.
Merocyanine 540, which only changes fluorescence by 1 part in 1000 in
response to a 90 mV MP change, has been shown to be sensitive to membrane
structural changes and has been used as an indicator of apoptosis on this
basis. If oxonols share the property, they may be useful as indicators of
apoptosis, but it won't be clear how much of the oxonol signal responds to
MP and how much to structural change.

As usual, nothing is simple.


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