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The most reliable method of producing mitochondrial loading of rhod-2 I have
found is the "cold load followed by warm incubation" procedure published by
Trollinger et al in 1997 (PMID:9245725). The warm incubate step is 37C,
without dye present for several hours and enables leakage of dye from the
cytosol while the dye trapped in the mitochondria is retained. The paper
states that cold loading alone does not give selective mitochondrial
loading. The externally added dye concentration should be as low as
possible - 1 micromolar or less. Mitochondria are very small compared to
the volume of the cytosol, so large concentrations of dye will just saturate
the mitochondrial uptake and end up in the cytosol and other unwanted
places. The borohydride reduction of rhod-2 AM is rarely effective in my
experience.
Also, I think you have to be careful about the doxorubicin treatment.
Doxorubicin is known to dissipate mitochondrial membrane potential which is
the driving force required for mitochondrial uptake and retention of rhod-2
AM.
Iain
Iain Johnson Consulting
Eugene, OR
On Tue, May 3, 2011 at 10:38 AM, Maria Fergusson
<
[hidden email]>wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hello,
>
> I would like to know if anyone has experience loading Rhod2 (calcium probe
> from Molecular Probes) specifically into mitochondria. I have been trying
> to load the dye into Hela and 3T3L1 cells using different temperatures(4
> degrees, RT and 37 degrees), concentrations (between 1-10uM), times (30
> min,1 hour) and we have treated the cells with doxorubicin (1-10UM) to see
> calcium influx. I added Pluronic 0.02% to load the Rhod2.
> I have simultaneously stained with Mitotracker to see mitochondrial
> localization.
> We consistently see a very strong Rhod2 staining that doesn't look
> mitochondrial.Some Rhod 2 is in the nuclei and some is in
> bright spots in a different compartment than mitochondria. We also tried
> reducing the dye with NaHB4.
> I would really appreciate the input of someone with experience loading Rhod
> 2 into mitochondria.
> Thank you
>
> Maria
>
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