ab113850 –

JC1 - Mitochondrial

Membrane Potential

Assay Kit

Instructions for Use

For the measurement of mitochondrial membrane potential by fluorescence plate reader This product is for research use only and is not intended for diagnostic use.

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Table of Contents

1. Introduction 3

2. Assay Summary 5

3. Kit Contents 6

4. Storage and Handling 6

5. Additional Materials Required 7

6. Assay Procedure 8

7. Data Analysis and Sample Data 13

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1. Introduction

This mitochondrial membrane potential (∆ψm) kit ab113850 uses

tetraethylbenzimidazolylcarbocyanine iodide (JC-1), a cationic dye

that accumulates in energized mitochondria. At low concentrations

(due to low ∆ψm) JC-1 is predominantly a monomer that yields

green fluorescence with emission of 530±15nm. At high

concentrations (due to high ∆ψm) the dye aggregates yielding a red

to orange colored emission (590±17.5nm). Therefore a decrease in

the aggregate fluorescent count is indicative of depolarization

whereas an increase is indicative of hyperpolarization. The

accompanying FCCP (carbonyl cyanide 4-(trifluoromethoxy)

phenylhydrazone) is an ionophore uncoupler of oxidative phos-

phorylation. Treating cells with FCCP eliminates mitochondrial

membrane potential and JC1 staining. JC1 is suitable for the

labeling of mitochondria in live cells and is not compatible with

fixation.

Membrane potential (∆ψm) is highly interlinked to many

mitochondrial processes. The ∆ψm controls ATP synthesis,

generation of ROS, mitochondrial calcium sequestration, import of

proteins into the mitochondrion and mitochondrial membrane

dynamics. Conversely, ∆ψm is controlled by ATP utilization,

mitochondrial proton conductance, respiratory chain capacity and

mitochondrial calcium. Hence pharmacological changes in ∆ψm can

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be associated with a multitude of other mitochondrial pathological

parameters which may require further independent evaluation.

Depolarization can be found in the presence of ionophores that could

induce nonselective cation channels or become selective mobile

ionic carriers. Protonophores such as FCCP and CCCP induce

reversal of the ATPase, as a compensatory mechanism that tries to

maintain ∆ψm, which will deplete ATP even in the presence of a

normal glycolytic pathway. Hyperpolarization could be found in the

presence of ATPase inhibition, inadequate supply of ADP, increased

supply of NADH, apoptosis due to oxidative stress and potentially

proton slippage due to cytochrome c oxidase dephosphorylation. In

either scenario, OXPHOS uncoupling ensues.

Limitations:

• FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC

PROCEDURES.

• Use this kit before expiration date.

• Do not mix or substitute reagents from other lots or sources.

• Any variation in operator, pipetting technique, washing

technique, incubation time or temperature, and kit age can

cause variation in binding.

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2. Assay Summary

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3. Kit Contents

• JC1: 0.5 mg

• 10X dilution buffer (sterile): 10 mL

• DMSO (cell culture tested): 1 mL

• 50 mM FCCP (Carbonyl cyanide 4-(trifluoromethoxy)

phenylhydrazone): 0.01 mL

4. Storage and Handling

Store all kit components at -20°C in the dark. Lyophilized JC-1 is

stable for 12 - 18 months if stored in the dark at -20°C. Once JC-1 is

reconstituted in DMSO, aliquot and store at -20°C.

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5. Additional Materials Required

• Fluorescence plate reader. The JC-1 aggregate can be

detected with similar settings to those used to detect

rhodamine (excitation/emission = 540/570nm) or texas red

(590/610nm).

• General tissue culture supplies

• PBS

• Fetal bovine serum

• Sterile, tissue culture treated, black 96-well microplates

• 50 – 300μL Multichannel pipettor

• Optional:

� Test compounds of interest.

� Uncouplers include CCCP (carbonyl cyanide 3-

chlorophenylhydrazone), 2’, 4’ Dinitrophenol

� 96-Well Deep Well Microplate with lids

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6. Assay Procedure

The protocol below is a guideline for measurement of

membrane potential with two specific cell lines. Use of different

cell lines will require optimization of protocol, in particular the

JC-1 concentration and the cell seeding per well.

A. Suspension cell culture and treatment (example human

HL60 cells)

1. Grow HL60 cells in glucose based media so that

approximately 2.5x107 cells are available on the day of the

experiment per plate

2. Make a 1X buffer solution as follows: 90mL of sterile

deionized water + 10mL of 10Xbuffer.

3. Make a 1X supplemented buffer solution as follows: 18mL

of 1X buffer solution + 2mL of FBS.

4. If performing toxicity assays, dilute compounds of interest in

1X supplemented buffer to 2X of final desired concentration

for the experiment. A 96-well deep well microplate may be

use in this step. Compounds may also be diluted in complete

media with 10% FBS without phenol red. Include positive

(100μM FCCP) and negative controls (vehicle of choice).

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5. Allow JC-1 and DMSO to warm to room temperature.

Reconstitute JC-1 in DMSO to generate a 1mM solution (mw

652).

6. Prepare JC-1 mix as follows: 10mL of 1X buffer solution +

10μL of 1mM JC-1 (final concentration 1μM).

7. Centrifuge the JC-1 mix at 13,000g for 3 minutes to

sediment non-soluble particles.

8. Collect cells and wash by centrifugation once in PBS.

9. Resuspend cells in 10mL of JC-1 mix and incubate at 37°C

for 30 minutes in the dark.

10. Wash cells by centrifugation with 10mL of 1X buffer solution

once

11. Resuspend 2x107 cells in 5mL of 1X supplemented buffer.

12. Seed a 96-well dark plate as follows: 200,000 stained

cells/50μL/well. Include blank wells (with non-stained cells).

13. If performing toxicity assays, add to each well 50μL of

previously diluted 2X compounds and treat for desired

period of time

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B. Adherent cell culture and treatment (example human HepG2

cells)

1. Grow HepG2 cells in standard media so that 3x106 to 4x10

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cells are obtained the day before the experiment.

2. Harvest cells the day before the experiment and seed a dark

96-well microplate with 15,000 cells per well. Allowed to

attach overnight.

3. On the day of the experiment, make a 1X buffer solution as

follows: 90mL of sterile deionized water + 10mL of 10X

buffer.

4. On the day of the experiment, make a 1X supplemented

buffer solution as follows: 18mL of 1X buffer solution + 2mL

of FBS.

5. Allow JC-1 and DMSO to warm to room temperature.

Reconstitute JC-1 in DMSO to generate a 1mM solution (mw

652).

6. On the day of the experiment, prepare JC-1 mix as follows:

10mL of 1X buffer solution + 200μL of 1mM JC-1 (final

concentration 20μM). Centrifuge the JC-1 mix at 13,000g for

3 minutes to sediment non-soluble particles.

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7. If performing toxicity assays, dilute compounds of interest to

the final desired concentration in 1X supplemented buffer

solution. A 96-well deep well microplate may be use in this

step. Compounds may also be diluted in complete media

with 10% FBS without phenol red. Include positive (100μM

FCCP) and negative controls (vehicle of choice)

8. Wash the HepG2 cells seeded on the 96-well plate with

100µL/well of PBS once.

9. Add 100μL/well of JC-1 mix and incubate for 10 minutes at

37°C in the dark. Include blank wells (with non-stained cells).

10. Wash the plate twice with 1X buffer solution.

11. If performing cytotoxicity assays, add compounds of interest

and treat for desired period of time.

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C. Fluorescence Reading

1. Set the fluorescent plate reader to perform an endpoint read.

2. Set excitation wavelength at 535 ± 17.5nm (aggregate

excitation only) or at 475 ± 20nm (for simultaneous

aggregate and monomer excitation)

3. Set emission wavelength at 590 ± 17.5nm (aggregate

emission only). If reading of the monomer species is also

desired, set a second emission reading at 530 ± 15nm.

4. FCCP 100μM treatment for 4 hours should decrease the JC-

1 aggregate signal to at least 25 – 30% from control levels.

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7. Data Analysis and Sample Data

Subtract background (A590 of non-stained cells) from test signal and

express as percentage from control. Data obtained with the JC-1

assay gives a relative measure of mitochondrial membrane potential

as a percentage of control and cannot be used for absolute

measurements of membrane potential in millivolts. Decrease in JC-1

signal may indicate either mitochondrial depolarization or cell death

and must be interpreted in parallel with a cytotoxicity assay (such as

the ATP detection kit MS957). The data in Figure 1 below shows the

uncoupling effect of FCCP acute treatment on HL60 cells as

measured with the JC-1 stain and read on a fluorescent plate reader.

m F

CCP

µµµµ

100

Veh

icle

contr

ol

0

5,000

10,000

15,000

Flu

ore

scen

ce C

ou

nts

[A590]

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Figure 1. Mitochondrial membrane potential assay result. Labeled

HL60 cells were seeded at 2x105 cells per well in glucose based complete

media and treated for 4 hours with 100µM FCCP and vehicle control

(DMSO). Cells were then transferred to a microplate and read on a

spectrophotometer. Mean and standard deviation is plotted for 3 replciates

from each condition.

This assay may be used for screening pharmacological

depolarization of mitochondria in any cell line. Depending on the

microplate template (see Fig. 2) either 3 or 4 compounds may be

tested in triplicate dose response per plate.

A.

Treatments

Component

A

Component

B

Component

C

Component

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

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Component

D

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B.

Figure 2. Suggested assay templates. Two assay examples are shown

above. The example on the top (A) allows for screening of four compounds

in dose response. Row A contains the diluent control to determine maximal

signal in the absence of compound. Row H contains non stained cells to

determine background fluorescence. The example (B) only allows for

screening of three compounds in dose response with perimeter wells as the

background fluorescence and column 2 as the diluent titration control

Column 1, column 12, row A and row H contain non-stained cells.

Treatments

Component

A

Component

B

Component

C

1 2 3 4 5 6 7 8 9 10 11

A

B

C

D

E

F

G

H

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Two assay examples are shown

for screening of four compounds

in dose response. Row A contains the diluent control to determine maximal

signal in the absence of compound. Row H contains non stained cells to

only allows for

of three compounds in dose response with perimeter wells as the

background fluorescence and column 2 as the diluent titration control.

stained cells.

Component

12

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