At 07:56 PM 2/24/97 +0000, you wrote:
>Anyone out there had experience with the mitochondrial membrane potential
>probe JC1 ?
>

Yep, I've been using it for over a year now. It is a difficult dye to use,
however the results can be fantastic.

I have also experienced the ppt forming in solution but I have found this is
dependent temperature. Allow me to give you a pick description of my loading
procedure.

To 500ul of buffer add 5ug of JC-1 (dissoved in DMSO). Incubate in a 30C
water bath, shaking for 10 mins. Then -slowly- add this soln to your cell
suspention. Incubate JC-1 suspention for a further 15 min in the water bath.
This is often the time that the ppt forms, if you load at room temperature.
Then spin down and resuspend your cells.

Photo-bleaching has only been a problem to me when I deviate from this
loading protocol.

Confocal details:
BioRAD MRC-1000, Ex = 488, Em collected at 530nm and 590nm simultaneously.
(Use the A1 and A2 blocks)

Calibration of mitochondrial membrane potential has been causing me
nightmares for quite some time. However, I have had some success (5 out of
15), that have worked.

Method:
I prepare a series of HEPES buffered solutions with increasing [K+], and
decreasing [Na+] (for osmotic equilibrium). I then add a K+ ionophore
(Valimomycin), to each solution. Once I've loaded the cells with JC-1, I
then add then to one of the K+ solutions. Then image.

Sorry if I'm a bit confusing. There are numerous (about 10) papers
discussing JC-1. If you like, I can send you a list of them.

Cheers,

Dave Bowser




_______________________________________________________________
David Bowser

CONfocal & Fluorescence Imaging Group (CON.F.I.G)
 Muscle and Cell Physiology Laboratory
  Department of Physiology
   University of Melbourne
    Parkville  VIC  3052
     Australia

Phone: 61-3-9344 5816
Fax:   61-3-9344 5818
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