This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4

Analysis of Mitochondrial Membrane Potential
with the Sensitive Fluorescent Probe JC-1

Andrea Cossarizza and Stefano Salvioli
Department of Biomedical Sciences
University of Modena School of Medicine
via Campi 287, 41100 Modena, Italy
phone +39 59 428.613
fax +39 59 428.623
E mail:






JC-1 staining


2.1 Materials
JC-1 fluorescent probe, plastic tubes for FACS analysis, complete medium, i.e. RPMI added with 10% fetal calf serum, PBS.
2.2. Methodology
1. Harvest cells (at least 2x105) from experimental samples, bring total volume up to 1 mL of fresh complete medium.

2. Stain cell suspension with 2.5 mg/mL JC-1. Shake cell suspension until the dye is well dissolved, giving a uniform red-violet color. To do this, it is also possible to vortex vigorously the suspension immediately after the addition of the probe.

3. Keep the samples in a dark place at room temperature for 15-20 minutes. The duration of the staining depends upon the cell type, but in our hands all the cells used (lymphocytes, cell lines of different origin, fibroblasts, keratinocytes, hepatocytes, etc.) responded quite well to the treatment. Wash twice centrifuging at 500 g for 5 min with a double volume of PBS.

4. Resuspend in 0.3 mL of PBS, then analyze immediatly with the flow cytometer, typically equipped with a 488 nm argon laser. Set the value of photomultiplier (PMT) detecting the signal in FL1 at about 390 V, and FL2 PMT at 320 V; FL1-FL2 compensation should be around 4.0%, while FL2-FL1 compensation around 10.6%. This is however the classical setting of the instrument we use in our laboratory, and it has to be taken into account that, as each instrument has a different sensitivity, a different setting can be necessary to obtain an optimal signal. Concerning instruments, the staining has been tested on several different apparatus such as an Excel, from Coulter (in Bergen, Norway), an Elite (Coulter) in Paris, some FACSCAN, a FACSTAR Plus and a FACSCalibur, from Becton Dickinson (in Krakow, Poland, or Modena and Venice, Italy), a Biorad Brite and a Partec (in Krakow too), and they work perfectly as well. Obviously, compensations have to be set in a different way.



3.1 Background information
The technique of JC-1 staining has been developed with the intent to detect DY in intact, viable cells. For this purpose JC-1 acts as a marker of mitochondrial activity, since the formation of J-aggregates, which give red emission, is reversible. Cells with high DY are those forming J-aggregates, thus showing high red fluorescence. On the other hand, cells with low DY are those in which JC-1 maintains (or re-acquire) monomeric form, thus showing only green fluorescence. Normally green fluorescence of depolarized cells is a little bit higher than that of polarized ones simply because of the presence of a higher amount of JC-1 monomers.


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Appendix 1: Stock solution:


JC-1 is dissolved in N,Ní-dimethylformamide (Sigma-Aldrich, cat. n. D8654) at the concentration of 2.5 mg/ml.

It is stored at -20°C. Light sensitive.


Appendix 2: Reagents

Molecular Probes,
Eugene, OR, USA
catalog No.: T-3168

Note: colture medium, saline solutions and washing buffers are depending on the cell type which is used for the experimental procedure (PBMC, fibroblasts, hepatocytes, etc.). For blood white cells, RPMI 1640 with 10% heat inactivated foetal calf serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine is normally used as complete colture medium.


Appendix 3: Equipment

Flow Cabinet TC60 Gelaire 
Flow Cytometer FACScan Becton Dickinson
Incubator CO2-AUTO-ZERO Heraeus 
Centrifuge Minifuge RF  Heraeus
Pipetman P20, P200, P1000 Gilson
Vortex Vibrofix VF1 Electronic Janke & Kunkel-Ika 

Appendix 4: Glossary


Mitochondrial membrane potential (Dy) is generated by mitochondrial electron transport chain, which drives a proton flow from matrix through inner mitochondrial membrane to cytoplasm, thus creating an electrochemical gradient. This gradient is in turn responsible for the formation of ATP molecules by F0-F1 ATP synthase. For this reason Dy is an important parameter for mitochondrial functionality and an indirect evidence of energy status of the cell.