That probe is expected to measure cytosolic Ca2+ movements but I found it several times in mitochondria.
Hello David,
Fura-2 AM, like its predecessor Fura-2, is known to stain also mitochondria (see paper here, for example: www.ncbi.nlm.nih.gov/pubmed/1383555). It is able to cross both outer and inner mitochondrial membrane and detect Ca2+ in the mitochondrial matrix. BAPTA-APM might be more specific for detecting cytosolic calcium (I have no first-hand experience with it, though).
Best regards!
Thanks! According to Kd of Fura-2 I am not sure that it could detect changes in mitochondrial calcium. My question is just for curiosity because sometimes, the staining of Fura2 in my cells is completly mitochondrial for 380
Hi David,
Could you check the loading conditions? Dyes like fura-2 AM if incubated with cells at 37ºC (during or after loading) will lose most of cytoplasmic fura-2. At 37ºC organic anion transporters of the plasma membrane work faster, extruding cytoplasmic fura-2. What remains after 1-2 hours at 37ºC is the dye trapped in organelles such as mitochondria.
Juan
The dye will load where there is hydrolysis (and net accumulation) and esterases are everywhere. Juan is right, a higher loading temperature favours compartmentation so always try room temp first.
As an aside, because of their different structures, different dyes compartmentalise to different extents (http://www.ncbi.nlm.nih.gov/pubmed/11032777), presumably because of substrate efficiency for esterases/transporters. If you want cytosolic-only, you might have to try a few different dyes for your cell type (clearly you might have to sacrifice using radiometric fura-2).
Maybe this helps:
If your cells (such as CHO cells) containing the organic anion-transports, probenecid (2–5 mM) or sulfinpyrazone (0.2–0.5 mM) may be added to the the dye working solution (final in well concentration will be 1-2.5 mM for probenecid, or 0.1 -0.25 mM for sulfinpyrazone) to reduce the leakage of the de-esterified indicators.
[http://www.aatbio.com/products/protocol/21010.pdf]
Cheers, Jörg
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