User Manual- ENZ-51019-KP002 Rev 2.1.1 September 2010.pub

Enabling Discovery in Life Science®
Mito-ID® Membrane Potential Cytotoxicity Kit
for microplates
Instruction Manual
Cat. No. ENZ-51019-KP002
For research use only.
Rev. 2.1.1 September 2010
for 2 x 96-well plates
Notice to Purchaser
The Mito-ID® Membrane Potential Cytotoxicity Kit is a member of the CELLestial® product
line, reagents and assay kits comprising fluorescent molecular probes that have been
extensively benchmarked for live cell analysis applications. CELLestial® reagents and
kits are optimal for use in demanding cell analysis applications involving confocal microscopy, flow cytometry, microplate readers and HCS/HTS, where consistency and reproducibility are required.
This product is manufactured and sold by ENZO LIFE SCIENCES, INC. for research use
only by the end-user in the research market and is not intended for diagnostic or
therapeutic use. Purchase does not include any right or license to use, develop or
otherwise exploit this product commercially. Any commercial use, development or
exploitation of this product or development using this product without the express prior
written authorization of ENZO LIFE SCIENCES, INC. is strictly prohibited.
Limited Warranty
These products are offered under a limited warranty. The products are guaranteed to
meet appropriate specifications described in the package insert at the time of shipment.
Enzo Life Sciences’ sole obligation is to replace the product to the extent of the purchase
price. All claims must be made to Enzo Life Sciences, Inc. within five (5) days of receipt
of order.
Trademarks and Patents
Enzo, CELLestial and Mito-ID are trademarks of Enzo Life Sciences, Inc. Several of
Enzo’s products and product applications are covered by US and foreign patents and
patents pending.
Contents
I. Introduction ............................................................... 1
II. Reagents Provided and Storage.............................. 2
III. Additional Materials Required ................................. 2
IV. Safety Warnings and Precautions........................... 3
V. Methods and Procedures ......................................... 3
A. CELL PREPARATION.......................................................... 3
B. REAGENT PREPARATION ................................................... 3
C. MEMBRANE POTENTIAL CYTOTOXICITY ASSAYS ................. 4
VI. Appendices ............................................................... 6
A.
FILTER SET SELECTION ................................................... 6
B.
RESULTS ........................................................................ 6
VII. References ................................................................ 9
VIII. Troubleshooting Guide ......................................... 10
I. Introduction
Mitochondria play a central role in diverse biological phenomena including
metabolism, bioenergetics, cancer and apoptosis. Recently, it has been
found that compromised membrane potential induced by drug accumulation in the mitochondria contributes to the toxicity of various organs, especially the heart and liver. Adverse drug reactions often remain undetected
until large numbers of patients have already been exposed. The ability to
test a compound’s safety at the stage of “lead drug selection” (10-100
compounds) would provide a valuable tool for drug screening research.
Enzo Life Sciences’ Mito-ID® Membrane Potential Cytotoxicity Kit measures mitochondrial membrane potential with a cationic dye that fluoresces
either green or orange depending upon mitochondrial membrane potential
status. In energized cells, the Mito-ID™ membrane potential dye exists as
a green fluorescent monomer in the cytosol and also accumulates as
orange fluorescent aggregates in the mitochondria. However, in cells with
compromised mitochondrial membrane potential, the Mito-ID® membrane
potential dye exists primarily as green fluorescent monomers throughout
the cytosol and no longer exhibits orange fluorescence in the mitochondria.
Compared with the commonly used cationic carbocyanine dye JC-1, the
Mito-ID® membrane potential dye offers greater solubility, better photostability, and higher sensitivity to mitochondrial membrane potential changes.
A control mitochondrial membrane potential perturbation agent, carbonyl
cyanide 3-chlorophenylhydrazone (CCCP), is provided for monitoring
changes in mitochondrial dynamics.
The Mito-ID® Membrane Potential Cytotoxicity Kit enables monitoring of
mitochondrial potential changes using a simple fluorescence microplate
reader. Potential applications of the kits are in preclinical drug safety
assessment using in vitro cell culture models to aid in the drug development process, and especially to differentiate among test compounds and
rank their order of potency.
1
II.
Reagents Provided and Storage
All reagents are shipped on dry ice. Upon receipt, the kit should be stored
upright and protected from light at ≤-20°C. When stored properly, these
reagents are stable for at least twelve months. Avoid repeated freezing
and thawing. The reagents provided in the kit are sufficient for 2 x 96well microplates.
Reagent
Quantity
®
Mito-ID MP Detection Reagent
200 µL
CCCP Control (2 mM)
100 µL
10X Assay Buffer 1
2.5 mL
50X Assay Buffer 2
0.5 mL
III. Additional Materials Required
 Fluorescence microplate reader or imaging plate reader with a filter set
or monochromator setting that provides Ex=490 nm/Em=570-590 nm
 96-well tissue culture microplate with black wall and clear bottom
 Calibrated, adjustable precision pipetters, preferably with disposable
plastic tips
 Glass microscope slides (optional)
 Glass cover slips (optional)
 Deionized water
 Anhydrous DMSO (optional)
 Serum (optional)
 Growth medium (e.g. Dulbecco’s modified Eagle medium, D-MEM)
2
IV. Safety Warnings and Precautions
 This product is for research use only and is not intended for diagnostic
purposes.
 Some components of this kit may contain hazardous substances.
Reagents can be harmful if ingested or absorbed through the skin and
may cause irritation to the eyes. Reagents should be treated as
possible mutagens and should be handled with care and disposed of
properly.
 Observe good laboratory practices. Gloves, lab coat, and protective
eyewear should always be worn. Never pipet by mouth. Do not eat,
drink or smoke in the laboratory areas. All blood components and
biological materials should be treated as potentially hazardous and
handled as such. They should be disposed of in accordance with
established safety procedures.
 To avoid photobleaching, perform all manipulations in subdued light
environments, in amber microcentrifuge tubes or protected from light by
other means.
V. Methods and Procedures
A. CELL PREPARATION
Cells should be maintained in 96-well microplates via standard tissue
culture practices. Seed cells in microplate(s) the day before test
compound/probe addition. The protocols described herein are based
upon Hela cells, for which it is highly recommended that cells in
culture medium are seeded on plates at a density of 2 x 104 to 3 x 104
cells per well at a seeding volume of 100 L per well. Keep the
microplate(s) in a humidified CO2 incubator overnight.
NOTE: 96-well black wall and clear bottom plates are highly recommended for this assay. Any cell number and plate coating requirements
should be optimized for the selected cell.
B. REAGENT PREPARATION
NOTE: Allow all reagents to thaw at room temperature before starting
with the procedures. Upon thawing, gently hand-mix or vortex the reagents prior to use to ensure a homogenous solution. Briefly centrifuge
the vials at the time of first use, as well as for all subsequent uses, to
gather the contents at the bottom of the tube.
3
1. Positive Control
CCCP (carbonyl cyanide 3-chlorophenylhydrazone) is a proton
ionophore and uncoupler of oxidative phosphorylation in
mitochondria. As such, it is useful for depolarizing mitochondria.
Addition of CCCP should cause a dose-dependent reduction in
mitochondrial orange fluorescence. Prepare this perturbation
agent as a positive control for mitochondrial membrane potential
loss. Before beginning the experiment, ensure that the vial of
CCCP Control has equilibrated to room temperature. Dilute the
CCCP Control (supplied as a 2 mM stock solution in DMSO) in
medium or buffer of choice to a final concentration optimized for
the cells being used. For HeLa or U2OS cells, it is recommended
to perform treatment with the agent using a final assay concentration of 2~4 µM CCCP in medium for about 15~30 minutes in order
to observe changes in mitochondria membrane potential changes.
Unused stock should be stored in aliquots at -20°C for about
6 months.
2. Mito-ID™ MP Dye Loading Solution
NOTE: The Mito-ID® MP Detection Reagent is light sensitive. Avoid
direct exposure of the reagent to intense light. Aliquot and store
unused reagent at -20°C, protected from light. Avoid repeated freeze/
thaw cycles.
For each 96-well plate, add 100 µL of Mito-ID® MP Detection Reagent, 1 mL 10X Assay Buffer 1 and 0.2 mL 50X Assay Buffer 2
into 8.7 mL deionized water. Mix well.
C. MEMBRANE POTENTIAL CYTOTOXICITY ASSAYS
I. Rapid Kinetics Measurement (for compounds that change
mitochondrial membrane potential within 30 mins)
1. Obtain prepared microplates containing cells (see section A).
2. Dispense 100 µL of the prepared Mito-ID® MP Dye Loading
Solution (see section B-2, above) directly into the growth
medium.
NOTE: It is NOT necessary to remove medium or wash the cells.
However, some media may result in high background levels, and
some test compounds may be sensitive to the presence of serum.
In these instances, growth media and serum should be removed.
Then, add 100 µL of the buffer of choice to the wells before adding
the dye loading solution.
3. Incubate the microplates for 30 mins at room temperature, or
37°C prior to addition of the test compound.
4
NOTE: The Mito-ID® MP dye is light sensitive. Be sure to protect
samples from light. The appropriate incubation time depends
upon the individual cell type and cell concentration used. Optimize
the incubation time for each experiment. DO NOT wash the cells
after dye loading.
4. Prepare the test compound by dissolving it in the buffer of
choice. The Mito-ID® Membrane Potential Cytotoxicity Assay
is optimized for compound addition at one-tenth of the final
well volume.
For positive control, dispense 20 µL of the provided CCCP
Control to a final concentration of 2~4 µM in medium or buffer.
NOTE: Titration of the CCCP Control may be required for optimal
results with different cell types.
5. Run the Mito-ID® membrane potential cytotoxicity assay by
monitoring the fluorescence at Ex=490 nm/Em=590 nm with a
fluorometric imaging plate reader or a fluorescence microplate
reader equipped with liquid dispensing capabilities. In live and
healthy cells, the mitochondria will fluoresce orange following
aggregation of the Mito-ID® MP dye. The orange J-aggregates
emit at 590 nm.
NOTE: Faster addition speeds can lead to better mixing of compounds and lower signal variance across the plate. Make sure to
follow the recommended experimental setup parameters provided
by the instrument manufacturer before reading the plate. It is also
important to run the signal test before the experiment. Different
instruments have their own intensity range.
II. Slow Kinetics Measurement (for compounds that change
mitochondrial membrane potential after more than 30 min
treatment)
1. Obtain prepared microplates containing live cells (see
section A).
2. Treat the cells with 10~20 µL of your compound under optimal
culture or buffer conditions for a time period sufficient for
assessing the effects of the compound.
For positive control, dispense 10~20µL of provided CCCP
Control to a final concentration of 2~4 µM in medium or buffer
for 15~30 minutes. Negative control cells should be treated
with the same vehicle (media or buffer) used to reconstitute or
dilute the inducer or inhibitor for an equal length of time under
similar conditions.
NOTE: It is NOT necessary to remove medium or wash the cells.
However, some media may result in high background levels, and
some test compounds may be sensitive to the presence of serum.
In these instances, growth media and serum should be removed.
Then, add 100 µL of the buffer of choice to the wells before adding
the dye loading solution.
5
NOTE: Titration of the CCCP Control may be required for optimal
results with different cell types.
3. Dispense 100 µL of the prepared Mito-ID® MP Dye Loading
Solution (see section B-2, page 4) into each well. Incubate for
30 minutes at room temperature or 37°C.
NOTE:
The Mito-ID® MP dye is light sensitive. Be sure to protect samples from light.
The appropriate incubation time depends upon the individual cell
type and cell concentration used. Optimize the incubation time for
each experiment.
4. Observe immediately with a fluorescent microplate reader or
imaging plate reader using an excitation setting of about 480
nm and an emission setting of about 590 nm. In live and
healthy cells, the mitochondria will fluoresce orange following
aggregation of the Mito-ID® MP dye. The orange
J
-aggregates emit at 590 nm.
NOTE: It is imperative that samples be analyzed immediately
following completion of staining. DO NOT wash the cells after
Mito-ID® MP dye loading. The fluorescence value in blank wells
added with the growth medium should be subtracted from the
values for those wells with cells treated with the test compounds.
VI. APPENDICES
A. FILTER SET AND MONOCHROMATOR SETTING SELECTION
The selection of optimal filter sets or monochromator settings for a
fluorescence microplate reader application requires matching the instrument optical specifications to the spectral characteristics of the
dyes employed in the analysis. Please consult your instrument or filter
set manufacturer for assistance in selecting optimal filter or monochromator settings.
Settings suitable for Texas Red or tetramethylrhodamine (TRITC) are
recommended for imaging the orange-fluorescent signal of the
Mito-ID® MP dye, indicating energized mitochondria.
B. EXPECTED RESULTS
In most eukaryotic cells the majority of ATP production is via oxidative
phosphorylation by the mitochondrial respiratory chain. Therefore,
the mitochondrial membrane potential makes up a large part of the
bioenergetic state of the cell and it is altered directly, depending upon
the cell’s energy needs. The ability to accurately measure mitochon6
drial membrane potential can give invaluable information about the
general health and function of the mitochondria, in particular of the
overall function of the respiratory chain and the potential of the
mitochondria to generate ATP and provide energy for other cellular
components. Measurement of the mitochondrial membrane potential
is useful in a wide variety of research areas and mitochondrial
dysfunction is implicated in diseases such as cancer, diabetes,
Parkinson’s disease, and stroke.
Typical results of fluorescence microplate-based analysis of cell
populations using the Mito-ID® Membrane Potential Cytotoxicity Kit
are shown in Figure 1. Using a conventional fluorescence microplate
reader, mitochondrial membrane potential is shown to decrease as a
function of CCCP concentration, as demonstrated by a decrease in
orange signal. The high Z’-factor (>0.9) obtained using this kit
demonstrates excellent signal-to-background ratios.
Figure 2 shows real time kinetic measurement of mitochondrial membrane potential changes with CCCP treatment using a conventional
fluorescence microplate reader with liquid dispensing capability for
test agent addition.
The Mito-ID® MP dye provides a more robust method to monitor mitochondrial membrane potential changes compared to JC-1, due to its
higher photostability, better aqueous solubility and higher sensitivity to
mitochondrial membrane potential changes. This kit offers true mixand-read capability and can be readily used for screening of large
compound libraries. 7
Figure 1. Using a conventional fluorescence microplate reader, mitochondrial membrane
potential was shown to decrease as a function of CCCP concentration, as demonstrated
by a decrease in orange signal. Enzo’s Mito-ID® MP dye is at least 10-fold more sensitive
to MMP loss than JC-1. The high Z’-factor (>0.9) obtained using the Enzo kit demonstrates excellent signal-to-noise and signal-to-background ratios. The error bars denote
the standard deviation of at least eight determinations.
Figure 2. Real time kinetic study of mitochondrial membrane potential changes arising
from CCCP treatment. Data were collected using a conventional fluorescence microplate
reader with liquid dispensing capability: Hela cells were seeded overnight in 25,000 cells
per 100 µl per well in a 96-well black wall/clear bottom plate. Next day, 100 µl of Mito-ID®
MP dye was directly added into each well and cells were incubated for 30 mins at room
temperature. CCCP (20 µl/well) was added using a BioTek dual liquid dispensor to achieve
concentrations of 1 µM. Orange Mito-ID® MP dye signal was shown to change in the
presence of CCCP as a function of time, as demonstrated by a decrease in signal, read
using the BioTek Synergy Mx fluorescence microplate reader.
8
VII. References
1. Smiley, Reers, Mottola-Hartshorn, Lin, Chen, Smith, Steele, and
Chen (1991) “Intracellular heterogeneity in mitochondrial membrane
potentials revealed by a J-aggregate forming lipophilic cation JC-1.”
Proc. Natl. Acad. Sci. USA 88: 3671-3675.
2. Cossarizza, Baccarani-Contri, Kalashnikova, and Franceschi (1993)
“A new method for the cytofluorimetric analysis of mitochondrial
membrane potential using the J-aggregate forming lipophilic cation
5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine
iodide (JC-1).” Biochem. Biophys. Res. Commun. 197 (1): 40-45.
3. Woollacott and Simpson. (2001) “High throughput fluorescence
assays for the measurement of mitochondrial activity in intact human
neuroblastoma cells.” J Biomol Screen 6(6): 413-420.
9
VIII. Troubleshooting Guide
Problem
Potential Cause
Suggestion
Fixed and/or permeabilized
cells fail to stain with the
mitochondrial membrane
potential dye.
This dye is only suitable for
live-cell staining.
Use the dye only for livecell analysis.
Stained cells have been
exposed to strong light.
Protect samples from exposure to strong light and
analyze them immediately
after staining.
Kit reagent has degraded.
Verify that the reagents are
not past their expiration
dates before using them.
Control cells without
treatment show low orange
signal.
Control cells are not healthy.
Extended storage of cells
after staining may adversely
affect their health. Use healthy cells.
Acquire data as soon as
possible after staining the
cells. Cell staining is too strong.
Mitochondrial membrane
potential dye is too concentrated for the cell type being
employed.
Dilute the staining solution
further with Assay Buffer.
Orange fluorescent mitochondrial membrane potential signal increases upon
treatment with the CCCP
Control.
The orange emission filter in
your instrument may be too
wide and green fluorescent
signal is being read, too.
Replace emission filter with
a narrower band pass filter.
Accuracy of data from
experiment to experiment is
poor.
The concentration of the
stained cell sample for data
acquisition is too low or
inconsistent between wells.
Repeat experiments using
a higher density of cells,
and carefully seed the wells
so that each well has the
same number of cells.
Poor staining observed.
10
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