The MitoPT TMRM Assay detects mitochondrial membrane depolarization utilizing the fluorescent dye TMRM. When accumulated in a negatively charge polarized mitochondria, TMRM fluoresces orange. When mitochondrial membrane potential collapses in apoptotic or metabolically stressed cells, TMRM reagent is dispersed through the cell cytosol and fluorescence levels drop dramatically. Analyze your results using a flow cytometer, fluorescence plate reader, or fluorescence microscopy.
- Prepare samples.
- Create controls with CCCP.
- Dilute 10X Assay Buffer 1:10 with diH2O.
- Reconstitute MitoPT TMRM with DMSO.
- Dilute MitoPT TMRM with 1X Assay Buffer.
- Add MitoPT TMRM to each sample.
- Incubate 15-30 minutes.
- Wash cells: add 1X Assay Buffer and spin cells.
- Remove supernatant and resuspend cells for analysis.
- Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. MitoPT TMRM excites at 548 nm and emits at 573 nm (orange).
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