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.
1
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
2
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.
accompanying
FCCP
(carbonyl
cyanide
The
4-(trifluoromethoxy)
phenylhydrazone) is an ionophore uncoupler of oxidative phosphorylation.
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)
mitochondrial processes.
is
highly
interlinked
to
many
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
3
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.
4
2. Assay Summary
5
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.
6
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 3chlorophenylhydrazone), 2’, 4’ Dinitrophenol
96-Well Deep Well Microplate with lids
7
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
7
approximately 2.5x10 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).
8
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
7
11. Resuspend 2x10 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
9
B. Adherent cell culture and treatment (example human HepG2
cells)
6
6
1. Grow HepG2 cells in standard media so that 3x10 to 4x10
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.
10
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.
11
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 JC1 aggregate signal to at least 25 – 30% from control levels.
12
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.
10,000
5,000
Ve
hi
cl
e
co
n
FC
tr
o
CP
l
0
10
0µ
m
Fluorescence Counts
[A590]
15,000
13
Figure 1.
Mitochondrial membrane potential assay result.
Labeled
5
HL60 cells were seeded at 2x10 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.
14
A.
Treatments
Component
A
Component
B
Component
C
Component
D
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
9
10 11 12
15
B.
Treatments
Component
A
Component
B
Component
C
A
B
C
D
E
F
G
H
1
2
3
4
5
6
7
8
9
10
11
12
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.
control
Column 1, column 12, row A and row H contain non-stained
stained cells.
16
17
18
UK, EU and ROW
Email: [email protected]
Tel: +44 (0)1223 696000
www.abcam.com
US, Canada and Latin America
Email: [email protected]
Tel: 888-77-ABCAM (22226)
www.abcam.com
China and Asia Pacific
Email: [email protected]
Tel: 108008523689 (中國聯通)
www.abcam.cn
Japan
Email: [email protected]
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
19
All information / detail is correct at time of going to print.