Hello,
Is the permeabilization step essential in Rhodamine 123 mitochondria stainingin order to measure the mitochondrial potential?
Do I need to dettach cells with Trypsin before staining?
Thanks in advance.
Hi Hamadi, Just saw your question about using Rhodamine 123, this can be loaded into adherent cells, just incubate for 45 min instead of 15 min. Also this cell permeant dye only loads into live cells with functioning mitochondria - so fixing cells would result in no Rhodamine 123 signal. If you need to fix the cells load MitoTracker Orange (575nm Em) or CMXROs (600nm Em) then fix the cells, these are the only 2 dyes that I know of that record & preserve the MitoTracker signal in fixed cells
Regards
Gary
Dear MadHi,
If you need to visualize a molecule located in the cell membrane (surface of cells), you do not need to permeabilise (ex: CD14). However, if you need to study a molecule inside the cell (cytoskeleton, Mitochondria…) permeabilization is needed in this case. You can fix your cells with PAF and permeabilise afte (Triton)r. If confocal imaging is used, cell observation could be performed without cell detaching by Trypsin.
Regards
Hi Hamadi, Just saw your question about using Rhodamine 123, this can be loaded into adherent cells, just incubate for 45 min instead of 15 min. Also this cell permeant dye only loads into live cells with functioning mitochondria - so fixing cells would result in no Rhodamine 123 signal. If you need to fix the cells load MitoTracker Orange (575nm Em) or CMXROs (600nm Em) then fix the cells, these are the only 2 dyes that I know of that record & preserve the MitoTracker signal in fixed cells
Regards
Gary
I would propose to perform a simple literature search using adequate sources (e.g. PubMed) to answer this question.
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