High-throughput measurement of mitochondrial membrane potentialin a neural cell line using a fluorescence plate reader

Alice Wong and Gino A. Cortopassi*

Department of Molecular Biosciences, University of California, 1311 Haring Hall, Davis, CA 95616, USA

Received 3 October 2002

Abstract

Mutations in mitochondrial genes cause mitochondrial genetic disease, which is often associated with deficiency of the mito-

chondrial membrane potential (MMP). We present a high-throughput method for measuring MMP in intact neural cells using

TMRM, a well-known potentiometric dye, in a 48-well plate format. Addition of known MMP depolarizing agents, FCCP or DNP,

resulted in a time- and concentration-dependent decrease in fluorescence, which was saturable, whereas the addition of drugs that

affect non-mitochondrial properties did not. A cell line deficient in mtDNA had decreased fluorescence, which was not further

depleted by a depolarizing agent. The high-throughput results are similar to those produced by more time-consuming and low-

throughput flow cytometry or microscopy methods. This plate-based system could facilitate the identification of cell-permeant small

molecules (i.e., drugs) that modify MMP, which could be used to enhance mitochondrial function, and also for screening small

populations of neural cells for mutations in nuclear or mtDNA genes that decrease MMP.

� 2002 Elsevier Science (USA). All rights reserved.

Keywords: High-throughput screen; TMRM; Mitochondria; mtDNA; Membrane potential; Apoptosis; Genetics; Rho-zero; Drug therapy

Mitochondria produce ATP through the process of

oxidative phosphorylation, which involves a series of

redox reactions that transfer electrons through multiple

protein complexes in the inner mitochondrial mem-

brane. As a result, protons are pumped out of the mi-

tochondria, generating the mitochondrial membrane

potential (MMP), which is not only harnessed to gen-

erate ATP, but is also responsible for mitochondrialCa2þ uptake [1,2], metabolite and protein transport

[3,4], production of reactive oxygen species [5,6], and has

also been related to the process of apoptosis [7–12].

Thus, the health and bioenergetic function of the mito-

chondria depend on its membrane potential.

Abnormal mitochondrial function has been attrib-

uted to cell death in several degenerative disorders in-

cluding Alzheimer�s and Parkinson�s diseases, as well astype 2 diabetes, stroke, and myocardial infarction

[13,14]. In addition, several debilitating diseases caused

by mutations in the mitochondrial genome have been

identified [15–17]. These mutations directly or indirectly

affect the electron transport chain, and thus, MMP.

Preservation of the MMP is essential during normal

conditions, and especially, during conditions of stress

and disease. Depolarization of the membrane results in a

reduction of ATP production and is also thought to

precipitate the release of pro-apoptotic factors in some

cell systems [7,10,11]. Furthermore, MMP is often defi-cient in mitochondria from patients with mitochondrial

genetic disease which is the result of mutations of the

mtDNA or of nuclear-encoded genes targeted to mito-

chondria. The phenotypes of such patients are pre-

dominantly neurological, i.e., preferentially affecting

neural and muscle cell types. Also, mitochondrial

oxidative stress has been invoked in several chronic,

progressive neurodegenerative diseases, including Alz-heimer�s, Parkinson�s, and ALS. Thus, there are goodreasons to search for therapeutic agents to increase

MMP in patients whose MMP is deficient, and for

therapeutic agents to decrease MMP as possible pro-

apoptotic drugs, for example, in the case of cancer.

Furthermore, there is no general assay for screening

Biochemical and Biophysical Research Communications 298 (2002) 750–754

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BBRC

* Corresponding author. Fax: 1-530-754-9342.

E-mail address: gacortopassi@ucdavis.edu (G.A. Cortopassi).

0006-291X/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

PII: S0006 -291X(02 )02546 -9

small populations of neural cells for nuclear or mtDNAmutations which decrease MMP. Thus there is a need

for high-throughput assays of living cells for drugs

which modify MMP and thus perforce mitochondrial

function. Several methods exist to measure MMP, many

of them using fluorescent dyes. All have their advanta-

ges and disadvantages. Besides measuring MMP, some

fluorescent dyes also measure the plasma membrane

potential, inhibit Complex I, or are photoreactive [18].We have developed an assay to measure MMP in

intact cells using tetramethylrhodamine methyl ester

(TMRM), a membrane-permeant, cationic fluorescent

dye that accumulates in the mitochondria according to

the Nernst equation [19–21]. When used at low con-

centrations, measurement of plasma membrane poten-

tial is minimal and the dye is the least toxic to

mitochondria [18,21–23]. The use of intact cells is ben-eficial, since permeabilization of the plasma membrane,

and potentially the mitochondrial membrane, can give

variable results. Using this assay, samples can be mea-

sured in a 48-well plate and respond to MMP-depolar-

izing agents.

Materials and methods

Cell culture. NT2 preneuronal and NT2 rho-zero cells were main-

tained in DMEM supplemented with 10% FBS, 50 lg/ml uridine, 1lMsodium pyruvate, penicillin, and streptomycin.

Measurement of mitochondrial membrane potential using the fluo-

rescence plate reader. Cells were isolated and washed in PBS before

resuspension in Hanks� buffered salt solution (HBSS) (1:0� 106 cells/ml) containing 50 nM TMRM. The cells were placed in a 48-well plate

and fluorescence was measured in a temperature controlled (37 �C)Cytofluor (PE Biosystems, Foster City, CA) plate reader for 3 cycles,

2min apart at 530–620nm. After the first 3 cycles, reagents (indicated

in the figure legends) were added to each well (indicated by an arrow)

and measurements were taken 2min apart for a total of 30min.

Background subtraction was conducted for each treatment and the

results shown are the means of three independent experiments per-

formed in triplicate. Error bars represent two standard error of the

mean.

Measurement of mitochondrial membrane potential by flow cytome-

try. Mitochondrial membrane potential was measured in digitonin

permeabilized cells using 50 nM TMRM [24]. Analysis was performed

on the FACSort flow cytometer (Becton–Dickinson, San Jose, CA),

with a 488 nm argon laser at the UC Davis Optical Biology facility.

Results and discussion

Known MMP depolarizers produce decreases in fluores-

cence in the high-throughput assay

A pharmacological proof that MMP was being

measured by the TMRM fluorescence was to use re-

agents known to abolish the membrane potential, FCCP

and DNP. After isolating cells and incubating them in

50 nM TMRM, fluorescence measurements were taken

every 2min for 3 cycles before the addition of depolar-izing agents. The addition of 10 lM FCCP or DNP

significantly depolarized the MMP compared to non-

treated cells (Fig. 1).

The decrease in fluorescence produced by depolarizers is

concentration-dependent and saturable, as expected of a

pharmacological depolarizer

The increase in TMRM fluorescence was time-de-pendent, as expected of an assay which requires the

transport of TMRM dye from the media to the mito-

chondria, and concentration of the TMRM dye by the

MMP. The decrease in fluorescence produced by known

MMP depolarizers was concentration-dependent and

saturable as expected of a pharmacological depolarizer.

Thus, low concentrations of DNP (0.01–1 lM) did notdecrease MMP as measured by the assay, whereas10 lM DNP decreased MMP strongly, and the depo-

larization produced by 100 lM DNP was no more than

that produced by 10 lM DNP, demonstrating satura-

bility of the process, as expected (Fig. 2). We did not

observe any hyperpolarization of MMP when giving the

neural cells oligomycin, an inhibitor of Complex V in

the electron transport chain, which in most cell types

causes a transient and small hyperpolarization of MMP,usually of 20mV or so, which is quite small relative to

the average total charge on the mitochondria, which

ranges between about 80 and 120mV physiologically

[25,26]. Other drugs, which are known to affect other

cellular processes (i.e., not MMP), did not cause a de-

crease of fluorescence in the assay more or less than

controls, in that neither cyclohexamide, a protein

Fig. 1. Collapse of mitochondrial membrane potential (MMP) by

addition of FCCP (N) or DNP (d). Fluorescence values were mea-

sured every 2min for 3 cycles before the addition (arrow) of FCCP,

DNP, or oligomycin. Measurements were then taken every 2min for a

total of 30min. MMP in cells receiving 10 lM FCCP or DNP did not

increase over time compared to untreated cells (j). Cells that received

oligomycin (r) had lower membrane potential as well.

A. Wong, G.A. Cortopassi / Biochemical and Biophysical Research Communications 298 (2002) 750–754 751

synthesis inhibitor, nor diltiazem, a calcium channelblocker, affected the TMRM fluorescence (Fig. 3).

MMP measurement in cells with a mitochondrial genetic

defect-rho-zero cells

Besides screening for drugs which alter MMP, an-

other potential use to which such a high-throughput

assay could be put is to screen cells for mitochondrial

genetic defects, which often produce decreases in MMP.To demonstrate this functionality, we chose to measure

MMP in NT2 rho-zero cells, i.e., cells that completely

lack mitochondrial DNA, and which have previously

shown to have little or no MMP [27,28]. NT2 rho-zero

cells had a significantly reduced MMP compared to

wild-type NT2 cells (Fig. 4), which was similar to that of

cells completely depolarized by DNP or FCCP; and the

addition of DNP did not further reduce the MMP as

measured by the assay (data not shown). Thus this assay

could potentially be used to screen clones or populations

of cells for those which have a mitochondrial geneticdefect that consequently reduces MMP.

Results of MMP measurement by high-throughput assay

and FACS are similar

As shown in Fig. 1, the addition of 10 lM DNP re-

duced the TMRM-dependent fluorescence in NT2 cells

(Fig. 5, top). As a comparison, we measured MMP using

flow cytometry (Fig. 5, bottom). Both methods show asimilar reduction in fluorescence when DNP was added

Fig. 3. Specificity of MMP. Addition (arrow) of cyclohexamide (d) or

diltiazem (N) did not affect MMP in NT2 cells (j).

Fig. 4. Measurement of MMP in genetically altered cells. Rho-zero

cells lack mitochondrial DNA and have a reduced MMP (N).

Fig. 5. Comparison of MMP, plate assay vs flow cytometry. The MMP

plate assay (top) and the flow cytometry method (bottom) resulted in

similar findings. After the addition of 10lMDNP, MMP decreased in

cells using either method.

Fig. 2. Concentration dependency of MMP. Increasing concentrations

of DNP were added (arrow) to NT2 cells. DNP (0.01–1lM) did notaffect membrane potential, while addition of 10 lMDNP (�) collapsedthe membrane potential. Supplementation of 100lMDNP (d) had no

additional affect.

752 A. Wong, G.A. Cortopassi / Biochemical and Biophysical Research Communications 298 (2002) 750–754

to the cells. The high-throughput plate assay gave similarresults as the flow cytometry method, confirming by a

known method the utility of this assay to measure MMP.

Summary and conclusions

We have developed a high-throughput, plate-based

assay of MMP in neural cells using TMRM, a known

mitochondrial potentiometric dye. The increase in

TMRM fluorescence is time- and concentration-depen-dent, which presumably affects the transport of the dye

from media to mitochondria, and concentration of the

dye by the MMP. The assay reliably detects decreased

fluorescence produced by agents which are known to

depolarize mitochondria specifically and the effects of

the drugs are concentration-dependent and saturable

(Figs. 1 and 2). Similarly, the assay detected no changes

in fluorescence induced by drugs which have no knownmitochondrial effect (Fig. 3). Thus this assay is a rela-

tively sensitive, fast, and specific screening method for

drugs or other small molecules that modify MMP.

Similarly, the assay detected decreased fluorescence in

cells with a deficiency of mitochondrial DNA, and

which are known to have a deficiency of MMP. Cur-

rently there are no general methods for rapid screening

(or selection) of colonies or small groups of mammaliancells for mitochondrial mutations. This genetic proof-

of-principle demonstrates that the assay could be used

to identify colonies or groups of cells with genetic

modifications of the mtDNA, and thus, could be used

for genetic screens of cells bearing deleterious mtDNA

mutations. A number of samples can be measured

concurrently using this rapid assay, and the dye used,

TMRM, is the least toxic to cells of the mitochondrialpotentiometric dyes [21] and can be incorporated into

mitochondria rapidly [19,29].

In summary, the simple and rapid method presented

specifically measures MMP in intact cells and could be

used to identify either drugs that affect mitochondrial

function, or as a screening method for colonies of cells

with mutations that alter mitochondrial properties, that

consequently decrease MMP.

Acknowledgments

We thank Dr. Heidi Gross for her helpful discussions and Carol

Oxford for her assistance with flow cytometry. This work was sup-

ported by USPHS Grant Ey12245.

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