Detection of mitochondrial membrane potential through JC1 staining

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shil22mishu
shil22mishu's picture
Detection of mitochondrial membrane potential through JC1 staining

In JC1 assay,  to quantitate mitochondrial membrane potential, the ratio of fluorescence reading in red channel to fluorescence reading in green channel is taken. So what is the reading we get for control condition , that is without any treatment? Is it always less than one or more than one, or can it be both ways? Either way , what does it indicate?
Does this ratio depends on cell type as well ? If so , what readings do we get generally for neurons and glial cells?

heehawmcduff
heehawmcduff's picture
The JC-1 dye measures

The JC-1 dye measures mitochondrial membrane integrity i.e. in healthy cells the dye aggregates in the mitochondria and fluoresces red, but in cells where the mitochondria have lost their membrane potential, the dye cannot aggregate and will stay in the cytoplasm and fluoresce green. 

Unfortunately, there is a very sensitive dose-dependent mechanism at play here, where the concentration of JC-1 incubated with the cells cannot exceed the amount that the mitochondria can hold or the dye will remain in the cytoplasm giving a false indication of mitochondrial membrane potential.

As a result, in answer to your first question, the dye can remain in the cytoplasm if 1) There is physically too much of it, or 2) the cells are unhealthy/apoptotic and the dye has flowed out of the mitochondria.  In my experience it is difficult to get an entirely 100% healthy population of cells where no green fluoresence will be observed but in certain cell lines, it is not unusual to have greater than 95% of the cells displaying only red fluoresence.

shil22mishu
shil22mishu's picture
Thanks for the reply , that's

Thanks for the reply , that's really helpful and informative. However , i need to know what are the raw values, people generally get , for the ratio of red /green in control condition, i.e. does it come less than 1, and if so can you site any paper for it.

The FFM
The FFM's picture
Here is a somewhat old review

Here is a somewhat old review.

If your search "jc-1 mitochondrial membrane apoptosis" There are 70 other papers that are open access which you can find via Pubmed that have data from JC-1 experiments that will help you get some comparisons for the fluorescence levels / ratios of the dye. 


Cytometry.
1999 Mar 1;35(3):181-95.

 
Analysis of apoptosis by laser scanning cytometry.
Bedner E, Li X, Gorczyca W, Melamed MR, Darzynkiewicz Z.
Brander Cancer Research Institute, New York Medical College, Valhalla, USA.
Flow cytometry techniques that are widely used in studies of cell death, and particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribute. These methods fail to recognize cell death lacking that attribute, as in some examples of atypical apoptosis. Since apoptosis was originally defined by morphologic criteria, we suggest that for any new cell system the cytometry-defined apoptosis be confirmed by morphologic examination. This quality assurance measure is now provided by laser scanning cytometry (LSC). LSC measurements of cell fluorescence are precise and highly sensitive, comparable to flow cytometry (FCM), and can be carried out on cells on slides, permitting cell by cell correlation of fluorescence cytometry with visual microscopic morphology. In this report we describe adaptations of various flow cytometry techniques for detection of apoptosis by laser scanning cytometry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is evidenced by high maximal pixel values for fluorescence of the DNA-bound fluorochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptosis include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC6(3)], and the aggregate dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The changes in plasma membrane phospholipids and transport function, also early in apoptosis, are probed by a combination of the fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-aminoactinomycin D. We also review methods of detection of apoptosis based on analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry makes it possible to discriminate those that are genuine from monocytes/macrophages that have ingested nuclear fragments via apoptotic bodies. Applications of flow cytometry and laser scanning cytometry in analysis of cell death are discussed and their respective advantages and disadvantages compared.
PMID: 10082299