MitoPT™-JC1

Assess mitochondrial permeability and depolarization events

ICT's original MitoPT™ JC-1 Mitochondrial Permeability Transition assay can be used to quantify apoptosis and mitochondrial depolarization. Non-apoptotic cells with polarized mitochondria will fluoresce orange-red; apoptotic cells containing depolarized mitochondria will fluoresce green.

Simple: Kit formats are easy to use. Just add the cell-permeant JC1 reagent to cell culture samples and incubate; no membrane permeabilization steps are necessary. Healthy cells will fluoresce orange-red, and depolarized cells will fluoresce green.

Fast: The MitoPT-JC1 Assay provides an assessment of the mitochondrial functionality status in less than 30 minutes.

Accurate: The potentiometric JC1 dye assumes a fluorescent green, monomeric form in mitochondrial compromised cells, providing a clear distinction between cells with polarized mitochondria and cells with depolarized mitochondria.

Quantitative: The MitoPT-JC1 Assay is compatible with flow cytometry, fluorescence plate reader, and fluorescence microscopy analysis methods.

MitoPT - JC1 Assay Principle

ICT's MitoPT™ JC-1 reagent is based on a lipophilic but slightly cationic fluorescent dye, commonly known as JC-1. The structure of this dye allows it to easily penetrate cells and healthy mitochondria. Once inside a healthy non-apoptotic cell, the potentiometric dye, bearing a delocalized positive charge, enters the negatively charged mitochondria where it aggregates and fluoresces red. These aggregates, first described by Jelley in 1937, are referred to as J-aggregates. When the mitochondrial collapses in apoptotic or metabolically stressed cells, the MitoPT JC-1 reagent can no longer accumulate inside the mitochondria. Instead, it is dispersed throughout the cell in a monomeric form, which fluoresces green. MitoPT™ JC-1 reagent excites at 488-490 nm. The monomeric dye structure emits at 527 nm, whereas the J-aggregates in polarized, healthy (non-apoptotic) mitochondria emit at 590 nm. Use of the MitoPT JC-1 assay kit allows the easy distinction between healthy/ non-apoptotic red fluorescent cells and apoptotic or mitochondrial membrane compromised green fluorescent cells.

The MitoPT™ JC-1 assay can be used in conjunction with your existing apoptosis or metabolic stress protocols. Grow your cells in culture and induce apoptosis or mitochondrial stress conditions according to your existing procedure. Once you have induced apoptosis or mitochondrial depolarization activity in your cells, add the 1X MitoPT™ JC-1 solution to each population and incubate the cells for an additional 15 minutes. During this incubation time, the JC-1 reagent will permeate the cells and their mitochondria. If the cell is not undergoing apoptosis or oxidative/metabolic stress, the mitochondrial membrane will remain intact, and the JC-1 reagent will concentrate to form J-aggregates and fluoresce orange-red. If the cell is apoptotic, the mitochondrial membrane potential will collapse, causing the JC-1 reagent to be dispersed throughout the cell in a monomeric form. In this monomeric form the JC-1 reagent will fluoresce green, giving the cells a green fluorescent color as well.

Analysis of JC-1

The MitoPT™ JC-1 assay can detect the mitochondrial depolarization event using three different technologies: flow cytometers; fluorometric plate readers; and fluorescence microscopes. The MitoPT™ JC-1 reagent excites at 488-490 nm. The monomeric dye structure emits at 527 nm, whereas the J-aggregates in polarized, healthy (non-apoptotic) mitochondria emit at 590 nm.
When cells stained with MitoPT JC-1 are evaluated on a flow cytometer, the instrument will measure the loss of by monitoring the reduction of red fluorescence in cell populations where the proton gradient across the inner mitochondrial membrane is diminished.
When evaluating JC-1 stained cells with a fluorescence plate reader, the instrument will again measure apoptosis induction or collapse by monitoring the reduction in the amount of red fluorescence output detected within the black 96 well plate wells. Healthy cells will give a high relative fluorescence unit (RFU) emissions output of red fluorescence. As the mitochondrial membrane potential collapses, indicating an apoptosis induction event, an increasing number of cells will give a lower red fluorescence RFU reading as the dispersed JC-1 dye coverts to a green fluorescent monomeric form.
Evaluation of JC-1 stained cells in fluorescence microscopy analysis will reveal non-apoptotic cells bearing orange-red fluorescent mitochondrial bodies. These mitochondrial organelles contain the fluorescent orange-red JC-1 dye aggregates within their healthy- polarized mitochondrial membranes. In contrast, apoptotic cells bearing an increasing number of depolarized mitochondrial bodies, will appear mostly green. Eventually, the entire cell will acquire a predominantly green fluorescence as the mitochondrial dissipates.

Following the flow cytometer and fluorescence microscope protocols, each sample to be stained requires only 0.5 mL of 1X MitoPT™ JC-1 solution (equal to 5uL of 100X MitoPT™ stock). The MitoPT™ JC-1 100 Assay Kit will test 100 samples; the MitoPT™ JC-1 400 Assay Kit will test 400 samples.  Following the fluorescence plate reader protocol, each sample requires 1 mL of 1X MitoPT™ JC-1 solution (requiring 10uL of 100X MitoPT™ JC-1 stock). The MitoPT™ JC-1 100 Assay Kit will test 50 samples in this way; the MitoPT™ JC-1 400 Assay Kit will test 200 samples.

MitoPT - JC1 Assay Kit (100 Tests, cat. #924)

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MitoPT - JC1 Assay Kit (400 Tests, cat. #911)

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Learn more at ICT's version 1.0 MitoPT webpage.

mitoPT jc1

Flow cytometer measurement of mitochondrial membrane depolarization in healthy and apoptotic Jurkat cells. Cells were treated with or without staurosporine for 3 hours at 37C and then labeled with MitoPT™ JC-1 for 15 minutes. Collapse of the mitochondrial membrane potential is indicated by an increase in the number of cells falling into R3 region, corresponding to a loss of red fluorescence, indicative of the onset of apoptosis or other depolarizing event.

Kit contains:

  • MitoPT-JC1 reagent
  • CCCP
  • Assay buffer

Sample staining protocol for suspension cells:

  1. Culture cells up to 1 x 10^6 cells/mL.
  2. Induce apoptosis or mitochondrial depolarization following your experimental protocol.
  3. Reconstitute the reagent with DMSO to form the stock concentrate (can be frozen for future use).
  4. Dilute stock concentrate with 1X Assay Buffer to form the working solution.
  5. Add 10mcL of the working solution directly to a 1mL aliquot of your cell culture for labeling.
  6. Incubate 10-15 minutes.
  7. Wash cells twice.
  8. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.