User Manual-ENZ-51011 - Rev 2.0.2 Nov 2010.pub

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Enabling Discovery in Life Science®
Total ROS Detection Kit
for fluorescence microscopy and flow cytometry
Instruction Manual
Cat. No. ENZ-51011
For research use only.
Rev. 2.0.2 November 2010
200 fluorescence microscopy assays
or 50 flow cytometry assays
Notice to Purchaser
The Total ROS Detection 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
This product is offered under a limited warranty. The product is 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 and CELLestial 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.............................. 1
III. Additional Materials Required ................................. 2
IV. Safety Warnings and Precautions........................... 2
V. Methods and Procedures ......................................... 3
A. REAGENT PREPARATIONS ..................................................... 3
B. CELL PREPARATIONS.............................................................. 4
C. FLUORESCENCE/CONFOCAL MICROSCOPY
(ADHERENT CELLS) ................................................................. 4
D. FLUORESCENCE/CONFOCAL MICROSCOPY
(SUSPENSION CELLS) ............................................................. 5
E. FLOW CYTOMETRY (ADHERENT CELLS) .............................. 6
F. FLOW CYTOMETRY (SUSPENSION CELLS) .......................... 6
VI. Appendices ............................................................... 7
A. FILTER SET SELECTION .......................................................... 7
B. SETTING UP OPTIMAL EXPOSURE TIME FOR
DETECTION OF THE DYE ........................................................ 7
C. ANTICIPATED RESULTS (FLUORESCENCE
MICROSCOPY) .......................................................................... 8
D. FLOW CYTOMETRY DATA ANLYSIS AND
ANTICIPATED RESULTS .......................................................... 8
VII. References .............................................................. 10
VIII. Troubleshooting Guide ......................................... 10
I. Introduction
Free radicals and other reactive species play influential roles in many
human physiological and pathophysiological processes, including cell
signaling, aging, cancer, atherosclerosis, macular degeneration, sepsis,
various neurodegenerative diseases (Alzheimer’s and Parkinson’s disease)
and diabetes. Once produced within a cell, free radicals can damage a
wide variety of cellular constituents, including proteins, lipids and DNA.
However, at lower concentrations these very same agents may serve as
second messengers in cellular signaling. Information-rich methods are
required to quantify the relative levels of various reactive species in living
cells and tissues, due to the seminal role they play in physiology and
pathophysiology. The Total ROS Detection Kit provides a simple and
specific assay for the real-time measurement of global levels of reactive
oxygen species (ROS), including peroxynitrite, in living cells.
This kit is designed to directly monitor real time reactive oxygen and/or
nitrogen species (ROS/RNS) production in live cells using fluorescence
microscopy and/or flow cytometry. The kit includes Oxidative Stress Detection Reagent (Green) as the major component. This non-fluorescent, cellpermeable total ROS detection dye reacts directly with a wide range of
reactive species, such as hydrogen peroxide, peroxynitrite and hydroxyl
radicals, yielding a green fluorescent product indicative of cellular production of different ROS/RNS types. The kit is not designed to detect superoxide and reactive chlorine or bromine species, as the fluorescent probe
included is relatively insensitive to these analytes. Upon staining, the
fluorescent product generated can be visualized using a wide-field fluorescence microscope equipped with standard green (490/525 nm) filter set, or
cytometrically using any flow cytometer equipped with a blue (488 nm)
laser.
II.
Reagents Provided and Storage
All reagents are shipped on dry ice. Upon receipt, the kit should be stored
at -20°C, or -80°C for long term storage. When stored properly, these
reagents are stable for at least twelve months. Avoid repeated freezing
and thawing.
Reagents provided in the kit are sufficient for at least 200 microscopy
assays or 50 flow cytometry assays using live cells (adherent or in
suspension.
Reagent
Quantity
Oxidative Stress Detection Reagent (Green)
ROS Inducer (Pyocyanin)
300 nmoles
1 µmole
ROS Inhibitor (N-acetyl-L-cysteine)
Wash Buffer Salts
2 x 10 mg
1 pack
1
III. Additional Materials Required
 CO2 incubator (37°C)
 Standard fluorescence microscope or flow cytometer equipped with a
blue laser (488 nm)
 Calibrated, adjustable precision pipetters, preferably with disposable
plastic tips
 5 mL round bottom polystyrene tubes for holding cells during induction
of ROS/RNS (for suspension cells only) and during staining and assay
procedure
 Adjustable speed centrifuge with swinging buckets
 Glass microscope slides
 Glass cover slips
 Deionized water
 Anhydrous DMF (100%)
IV. Safety Warnings and Precautions
 This product is for research use only and is not intended for diagnostic purposes.
 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 low light
environments or protected from light by other means.
2
V. Methods and Procedures
NOTE: Allow all reagents to warm to room temperature before starting with
the procedures. Upon thawing of solutions, gently hand-mix or vortex
thereagents 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.
A. REAGENT PREPARATIONS
Reconstitution or dilution of any and all reagents in DMSO should be
avoided, as this solvent inhibits hydroxyl radical generation in cells.
1. Detection Reagent
The Oxidative Stress Detection Reagent (Green) is supplied
lyophilized and should be reconstituted in 60 L anhydrous DMF
to yield a 5 mM stock solution. Upon reconstitution, the stock
solution should be stored at -20°C. Gently mix before use.
2. Positive Control
The ROS Inducer (Pyocyanin) is supplied lyophilized and should
be reconstituted in 100 µL anhydrous DMF to yield a 10 mM stock
solution. For use, a final concentration of 200-500 μM is recommended.
However, the optimal final concentration is celldependent and should be determined experimentally for each cell
line being tested. ROS induction generally occurs within 20-30
minutes upon pyocyanin treatment and may decrease or
disappear after that time. Plan accordingly.
3. Negative Control
The ROS Inhibitor (N-acetyl-L-cysteine) should be reconstituted in
123 L of deionized water to yield a 0.5 M stock concentration.
N-acetyl-cysteine is not readily soluble and may require vortexing.
For use, a final concentration of 5 mM is recommended.
However, the optimal final concentration is cell-dependent and
should be determined experimentally for each cell line being
tested.
Endogenous fluorescence of untreated cells should be determined
in advance or per assay.
4. 1X Wash Buffer
Prepare 1X Wash Buffer by dissolving the contents of the pack in
1 liter of deionized water. When not in use, the 1X Wash Buffer
should be stored refrigerated. Warm to room temperature before use.
3
5. ROS Detection Solution
Prepare the ROS Detection Solution as follows: To every 10 mL
of 1X Wash Buffer (see step 4) or culture medium, add 2 µL
Oxidative Stress Detection Reagent (Green). Gently mix.
To prepare smaller volumes of ROS Detection Solution, intermediate1:10 dilution of the Oxidative Stress Detection Reagent
(Green) in 1X Wash Buffer or culture medium is recommended.
B. CELL PREPARATIONS
Cells should be maintained via standard tissue culture practices.
Always make sure that cells are healthy and in the log phase of
growth before using them for the experiment.
C. FLUORESCENCE/CONFOCAL MICROSCOPY
(ADHERENT CELLS)
1. The day before the experiment, seed the cells directly onto glass
slides or polystyrene tissue culture plates to ensure ~ 50-70%
confluency on the day of the experiment.
IMPORTANT: Cells should be healthy and not overcrowded since
results of the experiments will depend significantly on the cells’
condition.
2. Load the cells with the ROS Detection Solution (see step A-5,
above) using a volume sufficient to cover the cell monolayer and
incubate under normal tissue culture conditions for 1 hour.
3. Carefully remove the ROS Detection Solution from the glass
slides by gently tapping them against layers of paper towel, or
from tissue culture plates. Optional: Cells may be washed with
the 1X Wash Buffer.
4. Treat the cells with an experimental test agent. Separate positive
control samples should be treated with the ROS Inducer
(Pyocyanin). Negative Control samples should be established by
treatment with the ROS Inhibitor (N-acetyl-L-cysteine).
NOTE: Cells should be treated with the ROS Inhibitor 30 minutes
prior to induction.
All treatments should be performed under normal tissue culture
conditions. It is recommended to perform a pretreatment by adding the ROS Inhibitor to the aliquots of ROS Detection Solution for
the last 30 minutes of the reagent loading. Treatment with an
experimental test agent or ROS inducer included with the kit
should be performed in the cell culture media without dye.
5. Carefully wash cells twice with 1X Wash Buffer in a volume
sufficient to cover the cell monolayer.
4
6. Immediately overlay the cells with a cover slip and observe them
under a fluorescence/confocal microscope using standard excitation/emission filter sets. Oxidative stress detection requires a filter
set compatible with Fluorescein (Ex/Em: 490/525nm). Make sure
prepared samples are protected from drying. Dried out cells may
present different fluorescence patterns.
D. FLUORESCENCE/CONFOCAL MICROSCOPY
(SUSPENSION CELLS)
1. Cells should be cultured to a density not to exceed 1 x 106 cells/
mL. Make sure that cells are in the log phase of growth before
starting an experiment.
IMPORTANT: Cells should be healthy and not overcrowded since
results of the experiments will depend significantly on the cells’ overall
condition. A sufficient volume of cells should be centrifuged at 400 x g
for 5 minutes, yielding a working cell count of 1 x 105 cells/sample.
2. Resuspend the cell pellet in 200 L of ROS Detection Solution
(see step A-5, page 4) and incubate under normal tissue culture
conditions for 1 hour with periodic shaking.
3. Centrifuge the cells at 400 x g for 5 minutes to remove the ROS
Detection Solution. Optional: Resuspend the cells in 5 mL
1X Wash Buffer, centrifuge them at 400 x g for 5 minutes and
remove the supernatant.
4. Treat the cells with an experimental test agent. Separate positive
control samples should be treated with the ROS Inducer
(Pyocyanin). Negative Control samples should be established by
treatment with the ROS Inhibitor (N-acetyl-L-cysteine).
NOTE: Cells should be treated with the ROS Inhibitor 30 minutes
prior to induction.
All treatments should be performed under normal tissue culture
conditions. It is recommended to perform a pretreatment by
adding the ROS Inhibitor to the aliquots of ROS Detection Solution for the last 30 minutes of the reagent loading. Treatment with
an experimental test agent or ROS inducer included with the kit
should be performed in the cell culture media without dye.
5. Centrifuge the cells at 400 x g for 5 minutes.
6.
Resuspend the cells in 5 mL of 1X Wash Buffer, centrifuge them
at 400 x g for 5 minutes and remove the supernatant.
7.
Resuspend the cells in 100 L of 1X Wash Buffer and apply a
20 L aliquot of the cell suspension, sufficient for 2 x 104 cells,
onto a microscope slide. Immediately overlay the cells with a
cover slip and analyze via fluorescence microscopy. Oxidative
5
stress detection requires a filter set compatible with Fluorescein
(Ex/Em: 490/525nm). Make sure that prepared samples are
protected from drying. Dried out cells may present different fluorescence patterns.
E. FLOW CYTOMETRY (ADHERENT CELLS)
1. The day before the experiment, seed the cells on appropriate tissue culture plates to ensure ~ 50-70% confluency on the day of
the experiment.
IMPORTANT: Cells should be healthy and not overcrowded since
results of the experiments will depend significantly on the cells’
condition.
2. Induce the cells with an experimental test agent. Separate positive control sample should be treated with the ROS Inducer
(Pyocyanin). Negative Control samples should be established by
treatment with the ROS Inhibitor (N-acetyl-L-cysteine)
NOTE: Cells should be pre-treated with the ROS Inhibitor at least
30 minutes prior to induction.
All treatments should be performed under normal tissue culture
conditions.
3. Remove the media with the inducers/inhibitors from the cells by
aspiration. Carefully wash cells twice with 1X Wash Buffer in a
volume sufficient to cover the cell monolayer, aspirate the supernatant.
4. Detach cells from the tissue culture plates using any appropriate
method, collect cells in 5 mL round-bottom polystyrene tubes and
wash them with 1X Wash Buffer. Centrifuge the cell suspension
for 5 min. at 400 x g at room temperature. Discard the supernatant.
5. Resuspend the cell pellet in 500 µL of ROS Detection Solution
(see step A-5, page 4). Stain cells for 30 min. at 37°C in the dark.
No washing is required prior to the analysis of the samples by flow
cytometry.
F. FLOW CYTOMETRY (SUSPENSION CELLS)
1. Cells should be cultured to a density not to exceed 1 x 106 cells/
mL. Make sure that cells are in the log phase of growth before
starting an experiment.
IMPORTANT: Cells should be healthy and not overcrowded since
results of the experiments will depend significantly on the cells’ overall
condition. A sufficient volume of cells should be centrifuged at
400 x g for 5 minutes, yielding a working cell count of 1-5 x 105 cells/
sample.
6
2. Induce the cells with an experimental test agent. A separate
positive control sample should be treated with the ROS Inducer
(Pyocyanin). A negative control sample should be established by
treatment with the ROS Inhibitor (N-acetyl-L-cysteine).
NOTE: Cells should be pre-treated with the ROS Inhibitor 30 minutes
prior to induction.
All treatments should be performed under normal tissue culture
conditions.
3. Centrifuge the cells at 400 x g for 5 minutes. Discard supernatatant.
4. Resuspend the cells in 5 mL of 1X Wash Buffer, centrifuge them
at 400 x g for 5 minutes and remove the supernatant.
5. Resuspend the cells in 500 μL of the ROS Detection Solution
(see step A-5, page 4) and incubate 30 min at 37°C in the dark.
No washing is required prior to the flow cytometry analysis.
VI. Appendices
A. FILTER SET SELECTION
For fluorescence microscopy, careful consideration must be paid to
the selection of filters. Dichroic filters should be selected in which the
“cut-off” frequency is optimally mid-way between the two emission
bands that are desired (one reflected, the other transmitted).
However, it is important to realize that dichroic filters have a somewhat limited reflectance range, i.e., a 600 nm short-pass dichroic filter
may actually reflect light <500 nm. When selecting filters, it is critical
to discuss with the filter or microscope manufacturer exactly what
wavelength specifications are required for both the transmitted and
the reflected light.
In addition, filters should be obtained that have the highest possible
transmission efficiency (typically requiring anti-reflection coating).
Each optic that an emission beam must traverse will remove some
fraction of the desired light. The difference between 80% transmission and 95% transmission for each detector may result in a greater
than three-fold difference in the amount of light available to the detector.
B. SETTING UP OPTIMAL EXPOSURE TIME FOR DETECTION
OF THE DYE
Optimal exposure times should be established experimentally for
each dye used in the experiment. Both negative and positive controls
should be utilized. Start with the negative control (untreated stained
cells) and set up the exposure time so the fluorescent background is
negligible. Then switch to a positive control (pyocyanin-treated cells)
and adjust the exposure time to record a bright fluorescent image.
7
Avoid saturation of the signal (very bright spots on the image). If
saturation of the signal occurs, decrease the exposure time. It is
recommended to acquire 5-6 single color images for each sample.
C. ANTICIPATED RESULTS (FLUORESCENCE
MICROSCOPY)
1. It is critical that positive (pyocyanin-induced) and control
(untreated) samples be included in every experiment for every cell
type. Negative (ROS Inhibitor-pretreated) sample is optional but
very helpful. In preliminary experiments, it is important to establish appropriate doses of inducers and inhibitors for each cell type
used.
2. Increased levels of oxidative stress give a uniform green cytoplasmic staining in the presence of the Oxidative Stress Detection
Reagent (Green).
3. ROS positive control samples, induced with ROS Inducer
(Pyocyanin), exhibit a bright green fluorescence in the cytoplasm.
4. Cells pretreated with the ROS Inhibitor (N-acetyl-L-cysteine)
should not demonstrate any green or orange fluorescence upon
induction.
5. Untreated samples should present only low autofluorescent background signal in any channel.
D. FLOW CYTOMETRY DATA ANALYSIS AND ANTICIPATED
RESULTS
1. It is critical that positive (pyocyanin-induced) and control
(untreated) samples be included in every experiment for every cell
type. Negative (ROS Inhibitor-pretreated) sample is optional but
very helpful. In preliminary experiments, it is important to establish appropriate doses of inducers and inhibitors for each cell type
used.
2. Cell debris should be gated out using FSC versus SSC dot plot.
3. Generate a log FL1 (X-axis) versus a log FL2 or SSC (Y-axis) dot
plot and add quadrants to it. Adjust quadrants so the majority of
control cells (80-90%) will fall into lower left quadrant. Keep the
same quadrant gate throughout the assay. Alternatively, log FL1
histogram can be used, where the mean fluorescence of the peak
for the untreated cells should fall within the first decade of a log
FL1 scale.
NOTE: Remember that different cell types demonstrate different
redox profiles therefore the number of the cells in the lower left
quadrant may vary significantly between the cell lines.
8
4. Cells with increased levels of oxidative stress demonstrate bright
green fluorescence in the presence of the Oxidative Stress Detection Reagent and will be detected using FL1 channel. Such cells
will appear in the two right quadrants of a log FL1 (X-axis) versus
FSC or SSC dot plot. If log FL1 histogram is used, the peak generated by the ROS positive cells will have increased FL1 fluorescence compared to a control cells’ fluorescence.
5. ROS positive control samples, induced with ROS Inducer
(Pyocyanin), exhibit bright green fluorescence and appear to be
positive in FL1 channel.
6. Cells pretreated with the ROS Inhibitor (N-acetyl-L-cysteine)
should not demonstrate significant green fluorescence upon
induction.
7. Control (untreated) samples should present only low autofluorescent background signal in any channel thus falling into the first
decade on a log FL2 scale.
Results of the experiments can be presented as percentage of the
cells with increased ROS production or as increase in the mean fluorescence of the induced samples versus control.
A
B
Figure 1. Jurkat cells were induced with 100 μM pyocyanin (general ROS inducer, panel A),
or 1 μM of t-butyl-hydroperoxide (peroxide inducer, panel B), stained with Total ROS Detection
Reagent and analyzed using flow cytometry. Untreated cells were used as a control. Cell
debris were ungated. The numbers in the inserts reflect the mean green fluorescence of the
control and treated cells.
9
VII. References
1. Tarpey, M. and Fridovich, I. Circ Res. 89 (2001), 224-236.
2. Batandier ,C., et al. J Cell Mol Med. 6 (2002), 175-187.
3. Gomes, A., et al. J Biochem Biophys Meth. 65 (2005), 45-80.
4. Wardman, P. Free Rad Biol Med. 43 (2007), 995-1022.
VIII. Troubleshooting Guide
Problem
Low or no fluorescent signal in positive control
Potential Cause
Suggestion
Dead or stressed
(overcrowded) cells
Prepare fresh cell culture for the
experiments. Make sure that the
cells are in the log growth phase.
Band pass filters are too
narrow or not optimal for
fluorescent probes
(fluorescence microscopy)
Multiple band pass filters sets provide less light than single band
pass ones. Check Methods and
Procedures section of this manual
and Appendix A for recommendations.
Insufficient fluorescent dye
concentration
Follow the procedures provided in
this manual.
Insufficient inducer concentration
Determine an appropriate concentration of inducer for the cell line(s)
used in the study.
Species of interest may
react with each other, thus
attenuating the expected
signal.
Check signaling pathways and all
the components present in the
cellular environment.
Make sure that time of detection is
optimized and the samples are
prepared immediately.
Inappropriate time point of
the detection
10
Green signal may quench if
concentration of product becomes
too high (due to long exposure to
the inducer). Otherwise, oxidized
product may eventually leak out of
the cells when left for a prolonged
period.
Problem
Potential Cause
Suggestion
Stressed (overcrowded)
cells
Prepare new cell culture for the
experiment. Make sure that the
cells are in the log growth phase.
Band pass filters are too
narrow or not optimal for
fluorescent probes.
High fluorescent
background
Wash step is necessary.
Follow the procedures provided in
this manual, making optional wash
steps mandatory.
Inappropriate time point for
detection
Make sure that time of detection is
optimized and the samples are
prepared immediately.
Inappropriate cell conditions
Inappropriate inhibitor
concentration (too low or too
high)
No decrease in the
fluorescent signal
after using a specific
inhibitor
Use correct filter for each fluorophore. Check Methods and Procedures section of this manual
and Appendix A for the recommendations.
Inappropriate time point for
detection
Make sure that you have viable
cells at the beginning of the
experiment, and that the inducer
treatment does not kill the cells
during the time frame of the
experiment .
Very low doses of inhibitor may
not affect ROS production by
inducer. Alternatively, very high
doses of the inhibitors may cause
oxidative stress itself and generate fluorescent signal. Optimize
the concentration of the inhibitor
and pretreatment time for each
particular cell line.
When cells are kept too long with
the inhibitors or at very high inducer concentrations, after a certain time, the inhibitor becomes
insufficient. Make sure that time
of detection is optimized.
Use correct filter for excitation and
emission. Check Methods and
Procedures section of this manual
Inappropriate filter set on the and Appendix A for the recommicroscope
mendations.
11
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