[Cytometry] TMRM

Mona Fares Mona.Fares at ki.se
Mon Feb 9 04:09:40 EST 2009

Dear friends,
 I want to use TMRM for mitochondrial membrane potential estimation for apoptosis assays by flowcytometry.
I need to know about the dye stability after thawing and if they are affected by frequent freezing and thawing. 
I want to carry out incubation in room tempreture because I found this will decrease the variation in tempreture during the whole experiment, but I need to know the appropriate time for incubation and the proper concentration. After washing the cells by the end of incubation do we need to resuspend them in PBS or flowcytometry fluid containing the same concentration of dye? I mean I want to know if the dye accumulation in the mitochondria is gradient dependent or constant?  
I found different protocol to make stock solution and working solution using different solvent as DMSO, Ethanol for stock solution and dH2O or HEPES buffer for working solution would you please help me to find the optimised method for making stock and working solution and how to freeze them?
Many thanks
Mona Fares
Research Fellow,Department of Laboratory medicine. Karoliniska Institute, Sweden 

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