MitoPT™- JC1 Assay Kit

Detect Mitochondrial Depolarization with MitoPT
As a member of the carbocyanine family of potentiometric dyes, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine I- / Cl- salt, commonly known as JC-1, is categorized as a slow membrane redistribution dye. The lipophilic structure of this dye allows it to easily penetrate cell and mitochondrial lipid bilayer membrane barriers. Once inside, JC-1 will redistribute itself within healthy polarized mitochondria in a Nernstian manner. As this dye accumulates within the negatively charged mitochondrial organelles, it reaches a saturation level at which J-aggregate formation occurs. This precipitated or aggregated form of the JC-1 dye fluoresces orange-red with blue light (488 nm) excitation. When the mitochondrial membrane potential collapses in apoptotic or metabolically stressed cells, the MitoPT™ JC-1 reagent will no longer concentrate within the mitochondria. Instead, it is dispersed throughout the cell in a monomeric form. In the monomer form, JC-1 will emit a green fluorescence upon excitation at 488 nm.

The MitoPT™ JC-1 assay can be used in conjunction with existing apoptosis or metabolic stress protocols. Each kit includes the JC-1 potentiometric dye reagent, a 10X wash buffer for removing any excess JC-1 dye after staining, and the generic mitochondrial membrane depolarizer Carbonylcyanide m- chlorophenylhydrazone (CCCP). JC-1 stained cells can be analyzed using flow cytometry, fluorescence microscopy, and fluorescence plate reader evaluation methodology.

Product Manuals:	MitoPT JC1 assay kit manual.pdf
Reagent Name:	JC-1

Sample Protocol:

1. Culture cells up to 1 x 10^6 cells/mL.
2. Expose cells to apoptotic inducing agent or metabolic stress environment using your experimental protocol.
3. Reconstitute the JC-1 reagent to form the stock concentrate (may be frozen for future use). Use 500 µL DMSO with the 100-test vial to make a 100X stock solution; use 1 mL DMSO with the 400-test vial to achieve a 200x stock solution.
4. Dilute the stock concentrate with 1X PBS, cell culture media, or wash buffer to form the working solution.
5. Remove cell culture supernatants by centrifugation or aspiration methods.
6. Add JC-1 working solution to your cell culture for labeling.
7. Incubate cells with JC-1 working solution, 10-15 minutes at 37° C in the dark.
8. Wash cells twice in 1x wash buffer.
9. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.

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4. Shirtliff, ME, et al. (2009) Farnesol-Induced Apoptosis in Candida albicans. Antimicrob. Agents Ch., 53: 2392 - 2401.
5. Weng, CJ, Yang, YT, Ho, CT, Yen, GC. (2009) Mechanisms of Apoptotic Effects Induced by Resveratrol, Dibenzoylmethane, and Their Analogues on Human Lung Carcinoma Cells. J. Agric. Food Chem., 57 (12): 5235-5243.
6. Hsu, CL, Yu, YS, Yen, GC. (2010) Anticancer Effects of Alpinia pricei Hayata Roots. J. Agric. Food Chem.,58(4): 2201-2208.
7. Sturrock, A, et al. (2010) Mechanisms of suppression of alveolar epithelial cell GM-CSF expression in the setting of hyperoxic stress. Am. J. Physiol.- Lung C., 298: L446 - L453.

Fluorescence microscope data

 Healthy cells fluoresce red, dying cells fluoresce green. Jurkat cells were stained with ICT’s MitoPT™-JC1 dye and analyzed with a fluorescence microscope containing a long band path filter (excitation at 490 nm and emission >510 nm). In non-apoptotic healthy cells (2 cells at left), the reagent aggregates inside intact mitochondria and fluoresces red. As the mitochondrial membrane potential drops and cells enter apoptosis, the dye disperses throughout the cell. The reagent then assumes a monomeric form and fluoresces green (3 cells on right).

 

  

Flow cytometry data

Using a flow cytometer to analyze cells labeled with MitoPT™-JC1, the instrument will measure apoptosis by monitoring the amount of red fluorescence in each region. Healthy cells with intact mitochondria fluoresce red due to aggregated MitoPT™-JC1 and appear in R2. As the mitochondrial membrane permeability collapses and cells enter apoptosis, MitoPT™-JC1 is dispersed throughout the cell where it converts to a monomeric form and fluoresces green. The amount of red fluorescence drops as these cells enter R3. In this example, Jurkat cells were either treated with DMSO (negative, non-induced cells) or with staurosporine (apoptotic, induced cells) for 4 hours and then labeled with MitoPT™-JC1 for 15 minutes.

 

 

 Fluorescence plate reader data

 

Using a fluorescence plate reader, healthy cells will give a high OD reading of red fluorescence; apoptotic cells will generate a lower reading of red fluorescence. In this example, Jurkat cells were either treated with DMSO (negative, non-induced cells) or with staurosporine (apoptotic, induced cells) for 4 hours and then labeled with MitoPT-JC1 for 15 minutes. As the mitochondrial membrane potential collapsed, indicating apoptosis, the amount of red fluorescence dropped by in half (51%) in Jurkat cells from 167 RFU to 82.

 

 

 

 

 

 

 

 

 

Target:	mitochondrial depolarization
Excitation / Emission:	488 nm / 527 nm and 590 nm
Method of Analysis:	Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader
Types of Samples:	cell culture, tissue
Kit Contents:	Kit #924: MitoPT JC-1 Reagent (one 100-test vial), 10x Assay Buffer (1 x 60 mL), 50 mM CCCP mitochondrial depolarizing agent (0.125 mL vial) Kit #911: MitoPT JC-1 Reagent (one 400-test vial), 10x Assay Buffer (2 x 125 mL), 50 mM CCCP mitochondrial depolarizing agent (0.6 mL vial)
Storage:	CCCP at -20° C, Other Kit Components at 2°-8° C, Ships Overnight (Domestic), International Priority Shipping
MSDS:	MitoPT JC-1 Reagent 10x Assay Buffer CCCP
Certificates of Analysis:	Inquire

Can I fix my cells before adding my JC-1 reagent?
No, fixation methods will most certainly cause a disruption in the proton pump-driven membrane potential gradient. This would cause all fixed cell populations to display a green JC-1 fluorescence indicative of cells bearing depolarized mitochondria.

Can I read my JC-1 stained cells the next day?
No, it is not a good idea to delay your analysis of mitochondrial membrane potential integrity. Cells that are stored for periods of time outside of their normal cell culture and 37° C environment will demonstrate a generalized reduction in mitochondrial membrane potentials over time. This would serve to invalidate any conclusions that could be drawn from your experiment.

How does the JC-1 dye assay differ from other commonly used potentiometric dye methods?
JC-1 resembles other commonly used potentiometric dyes in its manner of Nernstian redistribution within electrochemical gradients. Unlike the tetramethyl rhodamine dyes, which are also commonly used for this purpose, JC-1 displays bi-color fluorescence characteristics. Monomeric forms of the dye fluoresce green and the aggregated forms of the JC-1 dye, concentrated within polarized mitochondria, fluoresce orange-red.

Call 1-800-829-3194 for technical assistance or email Technical Support: help {at} immunochemistry.com.