Detection of Changes in Aym Using CMXRos Fixable

1. Harvest 1 x 105 cells and wash once in PBS.

2. Resuspend cells in 1 mL medium containing 30 mMCMXRos. (Stock solutions of CMXRos can be made in dimethyl sulfoxide at concentrations 100- to 200-fold more concentrated than required and stored at -20°C.)

3. Incubate cells at 37°C for 15 min.

4. Analyze cells using green fluorescence against cell numbers.

Alternatively, cells can be resuspended in medium containing 150 mM CMXRos, and after incubation they can be fixed by resuspending the CMXRos-stained cell pellet in 4% paraformaldehyde in PBS for 15 min at room temperature.

Cells can be stored at 4°C and/or stained for a second antigen of interest using membrane permeabilization procedures such as acetone, ethanol, or detergents.

Note: A positive control for these experiments is to treat cells with an uncoupling agent (e.g., 50 pMmClCCP), which completely abolishes the transmembrane potential.

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