Douglas Olson wrote: >We stain mitochondria (we've used mitotracker red) and then isolate them by a >simple cell lysis procedure which keeps them viable but removes them from the >cytoplasm. >Then, we sort to isolate pure mitochondria (to get rid of all the other >organelles and cell wall remnants). >However, the mitochondria don't seem to stay alive. We've considered that >they >may just get too beat up when sorted, but has anyone else tried a similar >expt. >(sorting organelles) and either failed or (even better) gotten it to work? What criteria do you use for mitochondria being "alive"? Most of the dyes which partition into mitochondria on the basis of membrane potential (rhodamine 123, cyanines such as JC-1 and DiOC6(3), and, presumably, the mitotracker dyes) attain high enough concentrations in the mitochondria to interfere with mitochondrial metabolism, so the organelles might not behave as would mitochondria isolated at lower purity (by centrifugation) without being exposed to dyes. Or are you looking at mitochondrial reproduction or DNA reproduction? I assume from the above that the mitochondria in the mixture you get after staining and lysing are functional, but the isolated mitochondria obtained by sorting aren't; if this is the case, there may be something they need that isn't present in whatever medium you sort them into. If the problem is with respiration and dye interference therewith, you might want to use a relatively water soluble dye which would be easier to wash out of the organelles after sorting. -Howard
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