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: cossariz@unimo.it

 
Introduction

The mitochondrial respiratory chain produces energy which is stored as an electrochemical gradient which consists of a transmembrane electrical potential, negative inside of about 180-200 mV, and a proton gradient of about 1 unit; this energy is then able to drive the synthesis of ATP, a crucial molecule for a consistent variety of intracellular processes. Several membrane permeable lipophilic cations, accumulated by living cells, organelles and liposomes exhibiting a negative interior membrane potential, have been used to study Dy. Such probes include those which exhibit optical and fluorescence activity after accumulation into energized systems, such as 3,3'-diehexiloxadicarbocyanine iodide [DiOC₆(3)], nonylacridine orange (NAO), safranine O, rhodamine-123 (Rh123) etc., radiolabelled probes, (i.e., [³H]methyltriphenyl-phosphonium, etc.) and unlabelled probes used with specific electrodes [i.e., tetraphenyl-phosphonium ion (TPP+) etc.]. These systems have several possible disadvantages, including the: a) time required to achieve equilibrium distribution of a mitochondrial membrane probe; b) degree of passive (unspecific) binding of probes to a membrane component, such as in the case of NAO, which detects mitochondrial mass as it binds to cardiolipin (9), or Rh123, which has several energy-independent binding sites (10), or DiOC₆(3) which, notwithstanding its high capacity to bind other membranes than those of mitochondria and its low sensitivity to agents capable of depolarize such organelles (11,12), has been widely used in the last years for studies on Dy; c) toxic effects of probes on mitochondrial functional integrity; d) sampling procedures; e) interference from light scattering changes and from absorption changes of mitochondrial components; f) requirement of large amounts of biological materials. TPP electrode affords an easy and precise tool to measure D y due to the: i) low interference between bound TPP+ and the membrane; and ii) lack of responses of the electrode to species different from TPP+. However, this method requires discrete amounts of biological samples and uptake of this lipophilic cation by intact mammalian cells is indeed a slow process.

To detect variations in D y at the single cell or at the single organelle level, a few years ago we have developed a new cytofluorimetric (FCM) technique by using the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) (13-15). JC-1 is more advantageous over rhodamines and other carbocyanines, capable of entering selectively into mitochondria, since it changes reversibly its color from green to orange as membrane potentials increase (over values of about 80-100 mV). This property is due to the reversible formation of JC-1 aggregates upon membrane polarization that causes shifts in emitted light from 530 nm (i.e., emission of JC-1 monomeric form) to 590 nm (i.e., emission of J-aggregate) when excited at 490 nm; the color of the dye changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized (16-18). Both colors can be detected using the filters commonly mounted in all flow cytometers, so that green emission can be analyzed in fluorescence channel 1 (FL1) and greenish orange emission in channel 2 (FL2). The main advantage of the use of JC-1 is that it can be both qualitative, considering the shift from green to orange fluorescence emission, and quantitative, considering the pure fluorescence intensity, which can be detected in both FL1 and FL2 channels.

Clearly, D y has been previously studied by flow cytometry, mostly by evaluating the changes in fluorescence intensity of cells stained with different, cationic dyes. Researchers used first Rh123 (19-21), then other molecules such as DiOC₆(3) (22). Typically, the signal coming from cells whose mitochondria had a low potential was much lower than that of control samples, and in a classical histogram depolarized populations go to the left. However, after the shift to the left the peaks (i.e. that of controls and treated cells) are not always perfectly separate, the operator has to decide "by eye" where the population of cells with depolarized mitochondria begins. These two fluorescent probes have this and other problems. Rh123 binding to mitochondria is difficult to calculate when the cell has a certain mitochondrial heterogeneity due, for example, to a high number of mature or immature organelles, as occurs in a continuously growing cell line. Moreover, different mitochondrial binding sites for Rh123 exist, i.e. sites which are freely accessible whatever the energy status of the mitochondria and sites which are hidden in the energized state and freely accessible in the deenergized form of the organelle. This has been attributed to different maturative states of the organelles. Thus, in a single cell, organelles can have different Rh123 binding sites with consequent different fluorescence emissions. As a result, it is very difficult to ascertain whether or not mitochondria bind Rh123 in an energy-dependent or energy-independent manner. However, the probe is perfect when used in association with propidium iodide, as this combination allows a clear and elegant distinction between dead and living cells (4).

DiOC₆(3) is more reliable for analysis of plasmamembrane potential rather than for studies on DY. Indeed, the first application of this probe in FCM was for the analysis of plasmamembrane potential (23). After this, DiOC₆(3) was used in isolated mitochondria to detect D y changes (24). Any cationic molecule goes to negative sites, and can be released when the negative charge decreases. If that molecule is fluorescent, the signal decreases when the membrane potential of the organelle is lost. Fluorescent molecules present in intact cells have a different behaviour. In our hands, DiOC₆(3) reacted properly when U937 cells were treated with FCCP, but such behaviour was not observed in cells treated with valinomycin. Moreover, when cells were kept in the presence of plasmamembrane depolarizing agents such as ouabain or high doses of extracellular K+, a consistent decrease in DiOC₆(3) fluorescence was noted, indicating a consistent sensitivity of the probe for plasmamembrane (12). This behaviour was not totally unexpected, as it is known that this probe can bind several membranes other than mitochondria, as also reported in the Handbook of Fluorescent Probes and Research Chemicals (edited by the Company that produces and sells this reagent, i.e. Molecular Probes, Eugene, OR, USA). Thus, using this probe, it is very difficult, if not impossible, to distinguish between depolarization of plasmamembrane or changes in DY in several physiological or pathological conditions, such as apoptosis, when both events can take place.

 

During their use, all reagents must be at room temperature and carefully checked for pH (7.4), since mitochondrial DY is very sensitive to alterations of both parameters.

Staining procedure must be carried under no direct intense light and incubation in the dark, because the light sensitivity of JC-1.

Always wear gloves when handling JC-1.

3.2 Critical Parameters

The need of high DY for the formation of J-aggregates makes this staining not suitable for fixed samples. Indeed, this technique has to be considered as a functionality test. Possible disadvantages come from the wide emission spectrum of the dye, which occupies two fluorescence channels, thus avoiding the use of other probes conjugated with FITC (e.g. monoclonal antibodies). The coupling with probes emitting in deep red detectable in FL3 channel is theoretically possible, but has many problems in compensating the different fluorescences, depending on the particular emission spectrum of each probe (quenching phenomenon). In particular, using propidium iodide for assessing cell viability in cells labelled with JC-1 can create consistent problems.

Only recently can the Authors test the stability of the probe in living cells fixed after the staining. This was done because cells were infected with HIV-1, and it is strongly recommended to fix such cells before running them into a flow cytometer. A light fixation with 0.5% formaldeide (few minutes at room temperature) however does not change the fluorescence pattern.

3.3 Trobleshooting

1. Presence of fluorescent molecules other than JC-1 in the sample: analyze first a non stained sample and set the instrument on its spontaneus fluorescence, then analyze the stained samples with the same setting. See also point 3.2.

2. Cells are not well stained: increase the amount of JC-1. Try to stain with an incubation at 37°C instead at room temperature.

3. Cell are too much stained: decrease the amount of JC-1. Leave the cells to stay a little bit longer in the JC-1 free PBS in order to allow the dye to reach the appropiate distribution equilibrium.

4. Fluorescence pattern too much widespread: see point 3.4. Do not consider any event with a very high FL2 fluorescence: very often they are JC-1 aggregates. Increase FSC threshold and discard debris with electronic gating: the presence of stained debris or broken cells can constitute a confounding element in the whole fluorescence pattern.

3.4 Anticipated results
It is recommended to perform each experiment using a "positive control" sample, in which mitochondria of all cells have been depolarized in order to have a correct setting of the instrument. Treating cells with drugs able to collapse DY, such as the K+ ionophor valinomycin (100 nM or more) or the proton translocator carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 250 nM), results in a dramatic change of the fluorescence distribution that indicates where depolarized cells have to go and helps a lot in setting the compensation. For problems related to intracellular trafficking and drug neutralization, valinomycin works much better than FCCP (and is also less expensive).

When the sample contains an heterogeneic cell population, it is possible to see different fluorescence patterns due to the variable content in membranes and mitochondria of cell subpopulations. It is typical the case of peripheral blood mononuclear cells (PBMC), formed by lymphocytes and monocytes, the first being smaller and with less mitochondrial content than the latter. Accordingly, the fluorescence pattern of JC-1 of such sample shows two distinct peaks, one corresponding to lymphocytes, and the second, brighter in both FL1 and FL2, corresponding to monocytes.

Another good control is that of mitochondrial mass, that can be done with nonyl acridine orange (NAO), that binds mitochondria independently of their energization state, and whose fluorescence is detectable in FL1. Typically, cells are incubated at the concentration of 0.5-1x10⁶ cells/mL with 10 µM NAO (Molecular Probes) for 10 min. in the dark at room temperature, washed twice in cold PBS and immediately analyzed. The result you obtain gives you an idea of the mass of mitochondria present within a cell, and allows you to be sure that the changes you see with a potential-sensitive probe are not dependent upon the simple loss of organelles. It is easy to imagine that also in this case monocytes are brighter than lymphocytes.

 3.5 Time considerations
The protocol of JC-1 staining does not require a long time (more or less 30 minutes). The duration of the staining procedure can obviously increase, depending on the number of samples to be analyzed.

3.6 Key references

JC-1 is dissolved in N,N’-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

 
 

JC-1 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  Labortechnik

 

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.