JC-1 Mitochondrial Membrane Potential Assay Kit

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Product Information

• Components : JC-1 dye 100 nmol×1vial, CCCP solution (10mM in DMSO) 500 μL×1vial

Description

• JC-1 is widely used in the observation of mitochondrial membrane potential, as it shows a characteristic fluorescence shift from green (530 nm) to red (590 nm) depending on the presence of a mitochondrial membrane potential. Mitochondrial membrane potential is an important facet of mitochondrial function and has been used as an indicator of cell health. In normal mitochondria, JC-1 collects and aggregates within the mitochondria. This aggregate fluoresces red. However, when the mitochondrial membrane potential is low, such as after having been depolarized, JC-1 cannot collect and aggregate within the mitochondria, leaving it as monomers with a green fluorescence. In this way, mitochondrial depolarization can be tracked through the red/green fluorescence intensity ratio, with smaller ratios indicating a larger extent of depolarization. To allow for comparison with depolarized cells as a control, this kit includes the depolarizing agent CCCP in a DMSO solution. JC-1 aggregates can be detected with filter sets designed to detect rhodamine (λex/λem = 540/570 nm). JC-1 monomers can be detected with filter sets designed to detect FITC (λex/λem = 485/535 nm).

Direction for Use

• Preparation of JC-1 DMSO stock solution (2mM)
• Add 50μL of DMSO to the included tube of JC-1 Dye and dissolve by pipetting.
• * Use prepared JC-1 DMSO stock solution within 1 month.
• 
  
• Preparation of JC-1 working solution
• First, add an appropriate amount of the JC-1/DMSO stock solution to the bottom of a microtube. Add an appropriate amount of medium and mix by pipetting. Adjust so the final concentration of JC-1 is in the range of 1-15 μM.
• * If the fluorescence signals are weak, optimization of JC-1 concentration and/or incubation time may be necessary.
• *When preparing JC-1 working solution, it is imperative that medium be added to the JC-1/DMSO stock solution. Reversing the order greatly increases the chances of JC-1 precipitating.
• *Use the JC-1 working solution within 1 day.
• 
  
• Preparation of CCCP working solution
• Add an appropriate amount of CCCP/DMSO stock solution (10mM) into a microtube. Add an appropriate amount of medium and mix by pipetting. Adjust the final concentration of CCCP to fall within the range of 5-100 µM.
• * If mitochondrial membrane depolarization is not observed, optimize the concentration of CCCP in working solution and/or incubation time.
• * Use the CCCP working solution within 1 day.
• 
  
• Mitochondrial membrane potential determination using JC-1

1. Seed cells into a dish or a chamber slide and incubate at 37°C in a 5% CO₂ incubator.
2. Prepare a control sample by applying CCCP. Incubate the cells at 37°C in a 5% CO₂ incubator for 30–90 minutes.
3. Remove half of the medium from each well (both samples and CCCP controls). Add to each well an equivalent amount of 2x JC-1 working solution.
   * For example, if the final concentration of JC-1 is to be 2 μM, apply a 4 μM JC-1 working solution to wells.
4. Incubate the cells at 37°C in a 5% CO₂ incubator for 20–60 minutes.
5. Remove medium from wells, and wash 3x with PBS.
6. After the final wash, observe the cells under a fluorescence microscope, or alternatively, measure fluorescence intensity via a plate reader.

Storage

• Store at -20°C. This product is photosensitive and should be protected from light. Use within 6 months.