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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