The MitoPT JC-1 Assay detects mitochondrial membrane depolarization utilizing the fluorescent dye JC-1. When accumulated in a negatively charged polarized mitochondria, JC-1 fluoresces orange. When mitochondrial membrane potential collapses in apoptotic or metabolically stressed cells, JC-1 reagent is dispersed through the cell and will fluoresce green. 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 JC-1 with DMSO.
- Dilute MitoPT JC-1 with 1X Assay Buffer.
- Add MitoPT JC-1 to each sample.
- Incubate 15-30 minutes.
- Wash cells: add 1X Assay Buffer and spin cells (twice).
- Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. MitoPT JC-1 excites at 488 nm. Aggregated MitoPT JC-1 (orange) emits at 590 nm, monomeric MitoPT JC-1 (green) emits at 527 nm.
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