Product Information Sheet

MarkerGeneTM Live Cell Fluorescent

Reactive Oxygen Species Detection Kit
Product M1049

Marker Gene Technologies, Inc.

University of Oregon Riverfront Research Park
1850 Millrace Drive
Eugene, Oregon 97403
1-888-218-4062
www.markergene.com

MarkerGeneTM Live Cell Fluorescent

Reactive Oxygen Species Detection Kit
(Product M1049)
2

NOTE: The following information is given as a viable methodology for use of the
MarkerGeneTM Live Cell Fluorescent Reactive Oxygen Species Detection Kit. Users
may determine their own best conditions for use dependent on the specific conditions
present in their experiment.

I. OVERVIEW
In healthy aerobic cells, ROS generation occurs at a controlled rate but under
high stress conditions, its production is greatly increased, resulting in changes of
many cell components including proteins and lipids.
Fluorogenic peroxidase substrates are converted to highly fluorescent products
in the presence of the enzyme and hydrogen peroxide but can be relatively
unstable for use in enzyme-linked immunosorbent assays (ELISAs). By forming
the diacetate derivates, intracellular applications are more efficient, whereupon
the acetates are cleaved by endogenous esterases, releasing the intact
substrate. In the presence of nonspecific ROS, commonly produced during
oxidative stress, the fluorescein substrate becomes oxidized, emitting green
fluorescence.
The MarkerGeneTM Live Cell Fluorescent Reactive Oxygen Species Detection Kit
utilizes the cell permeable substrate, 2',7'-dichlorofluorescin diacetate (Product
M0807), a reliable fluorogenic marker for ROS detection. Upon enzyme activity,
the highly fluorescent dye, 2',7'-dichlorofluorescein is produced, with EX: 495nm
and EM: 530 nm. In addition, this kit also contains t-butyl hydroperoxide, a
common ROS inducer, as a positive control. The assay kit contains enough
reagents to prepare up to 250 mL of staining solution, or approximately 100
assays (12-well tissue culture plate format), or 100 x 96-well tissue culture
microtiterplate staining assays. Methods for testing various drug or inducer
compounds are also described. This assay can be adapted for high-throughput,
high-content assay screening analyses.
Technical assistance is available online at techservice@markergene.com or by
telephone at 1-888-218-4062 during our regular business hours.

II. MATERIALS
A.)

Fluorescent Substrate Reagent: Five vials containing 500g

each of 2',7'-Dichlorofluorescin Diacetate (Product M0807).

B.)

ROS Inducer: 500 L of t-butylhydroperoxide (TBHP) 7.77M

solution in water.

C.)

Buffer Solution: 1mL Dimethylsulfoxide (DMSO).

Storage and Handling: Store kit at 2-6C upon receipt. Do not freeze the tbutylhydroperoxide solution. 2', 7'-Dichlorofluorescin diacetate should be handled with
care and after removing the vials for use, quickly return remaining vials to bag and
reseal. Solutions of 2',7'-Dichlorofluorescin diacetate in DMSO can be stored at -20C.
In case of contact with skin or eyes, wash thoroughly with soap and cold water.
Reagents should be stable for at least 6 months following purchase. High background
fluorescence readings for blank samples will indicate decomposition.

III. PROTOCOL FOR HTS TIMED ASSAY FORMAT **

Adherent cells:
1) Plate adherent cells in 96-well tissue culture plates at a density of
25,000 cells / well and allow to stabilize for 24 hours before the assays.
2) Prepare a 10mM solution of the Fluorescent Substrate Reagent, 2',7'Dichlorofluorescin diacetate, by adding 100L of DMSO to one vial of
2',7'-Dichlorofluorescin diacetate (500g) and gently vortexing until solid
is dissolved.
3) Dilute this substrate solution to a 20M working solution by adding 8L of
the 10mM 2',7'-Dichlorofluorescin diacetate solution to 4mL of warm DPBS, HBSS/Ca/Mg or other suitable buffer or into media. Alternately
dissolve the entire 10 mM substrate solution prepared in Step 2 with 50
mL of buffer or media as above. NOTE: This solution is unstable and
must be used immediately for staining cells in culture.
4) Load cells with the resulting 20M working substrate solution for 30-45
minutes (200 L per well for a 96-well format).
5) Remove the substrate solution by vacuum suction, wash with HBSS and
replace with HBSS containing 10% serum before treatment with ROS
inducing agents, or TBHP , 25 -100 M (as a positive control) at

concentrations appropriate concentration to generate ROS activity for

between for 1-4 Hours.
6) Place plates in a fluorescence microplate reader** and record both end
point and area-scan readings at 4 hours. Alternately, kinetic readings can
be recorded at 30 minute intervals to determine an optimum end point
time for the assay. Read plates at 485nm with emission at 528nm.
Non-adherent cells:
1) Grow non-adherent cells in culture flasks to a density of 6 x 105cells/mL
2) Centrifuge and resuspend the cell pellet in 10 mL HBSS.
3) Prepare a 10mM solution of the Fluorescent Substrate Reagent, 2',7'Dichlorofluorescin diacetate, by adding 100L of DMSO to one vial of
2',7'-Dichlorofluorescin diacetate (500g) and gently vortexing until solid
is dissolved. NOTE: This solution is unstable and must be used
immediately for staining cells in culture.
4) Add 20L of the 10mM substrate solution prepared in Step 3 to the
suspended cells to give a final concentration of 20M working solution of
the 2,7 dichlorofluorescin diacetate. Incubate for 30-45 minutes.
5) Centrifuge and resuspend cells in HBSS buffer containing 10% serum
(approx. 1 mL). Count the cells using a hemocytometer or other method,
and plate out the substrate-loaded cells at 100,000 cells / well in 96-well
plates for use in the assay.
6) Treat the cells with the test ROS inducing agents or TBHP (as a positive
control, at concentrations between 25 -100 M) for up to 4 Hours.
7) Place plates in a fluorescence microplate reader** and record both end
point and area-scan readings at 4 hours. Alternately, kinetic readings can
be recorded at 30 minute intervals to determine an optimum end point
time for the assay. Read plates at 485nm with emission at 528nm.

** Note: Assays were run using the BioTek monochromator-based Synergy Mx

Multimode Plate Reader. Consult your instrument manual or contact the manufacturer
for specific parameters for your instrument. Fluorescence can be observed using
standard fluorescein filter sets for fluorescein-based fluorophores. See the table below
or visit our website for a list of common fluorescence filter sets for use with 2,7dichlorofluorescein (http://www.markergene.com/Filter Sets.htm).

IV. PROTOCOL FOR FLUORESCENCE MICROSCOPY

1.)

Fluorescence can be observed using standard fluorescein filter sets for

fluorescein-based fluorophores. See the table below or visit our website
for a list of common fluorescence filter sets for use with 2,7dichlorofluorescein (http://www.markergene.com/Filter Sets.htm).

2.)

Culture adherent cells in 60mm, 100 mm or 12-well tissue culture plates

onto several sterile glass coverslips until acceptable cell densities are
reached (usually grown for 1-3 days prior to use, 60-70% confluency).
Culture non-adherent cells in tissue culture flasks until acceptable cell
densities are reached (approx. 6 x 105cells/mL).

3.)

Add drugs or test samples to the cells at various concentrations during this
growth period. Skip to step #6 if induction of ROS with the standard tbutyl hydroperoxide is not desired.

4.)

Positive Control-Induction of ROS: Prepare a solution of the ROS

Inducer Reagent by first adding 2L of TBHP (7.77 M) to 154L of water
to make a 100 mM working solution, slowly pipeting up and down to mix.
Then dilute in appropriate growth media to give a final concentration of
100M (Example: add 5L of the 100mM TBHP solution per 5 mL of
media). NOTE: Media can contain serum (up to 10%) during this step.

5.)

Apply sufficient 100M t-butyl hydroperoxide (TBHP) working solution in

media to cells adhering to cover slips, or collect non-adherent cells by
gentle centrifugation (~1000g) and resuspend in working solution.
Incubate at 37C/10%CO2 (standard incubation period is 60-90 minutes
but may vary depending on cell line). After incubation, label cells with
2',7'-Dichlorofluorescin diacetate as described in the following steps.

6.)

Prepare a 10mM solution of the Fluorescent Substrate Reagent, 2',7'Dichlorofluorescin diacetate, by adding 100L of DMSO to one vial of
2',7'-Dichlorofluorescin diacetate (500g) and gently vortexing until solid
is dissolved.

7.)

Dilute this substrate solution to a 25M working solution by adding 10L of

the 10mM 2',7'-Dichlorofluorescin diacetate solution to 4mL of warm DPBS, HBSS/Ca/Mg or other suitable buffer or into media. Alternately
dissolve the entire 10 mM substrate solution prepared in Step 6 with 40
mL of buffer or media as above. NOTE: The working substrate solution is
unstable, and must be used immediately for staining cells in culture.

8.)

Remove media (or media containing ROS inducer Reagent or Drug

samples) from adherent cells by suction. Gently wash adherent cells with
warm PBS. Media can be removed from non-adherent cells using gentle
centrifugation (~1000g) to collect cells and decanting supernatant. Nonadherent cells can be washed by suspension in warm PBS followed by
centrifugation to collect cells.

9.)

Apply 25 M 2',7'-Dichlorofluorescin diacetate solution, enough to cover

cells adhering to coverslips. Resuspend non-adherent cells in the 2',7'Dichlorofluorescin diacetate solution.

10.)

Incubate for 30-45 minutes while protected from light.

11.)

If desired, counterstain with 1M Hoechst 33342 (EX/EM: 350/460nm)

(add 1L of 1mM Hoechst 33342 per 1mL of 25M 2',7'-Dichlorofluorescin
diacetate solution) by adding to the 25M 2',7'-Dichlorofluorescin diacetate
solution during the last 5 minutes of incubation (The Hoechst 33342 is not
provided with this kit).

12.)

After incubation, gently wash cells three times with warm D-PBS or serumfree media (@ 37oC). Adherent cells on coverslips can be bathed in wells
containing the wash solution. To wash non-adherent cells, centrifuge at
low speed (~1000g) to collect cells and resuspend in wash solution.

13.)

Mount* and view the cells using an appropriate fluorescence microscope

(if cells were also labeled with Hoechst, use filter sets appropriate for
DAPI to locate cells on coverslip, followed by fluorescein filter sets for cell
imaging.).

14.)

Adjust the photographic time and F-stop values to reduce background

illumination, and improve contrast, or use an appropriate method for digital
photography contrast optimization.

*Adherent cells can be viewed by inverting stained coverslips onto a clean

microscope slide containing a drop of media or D-PBS. Optionally, a latex slide
gasket (Immuno-Cell, Belgium, www.immuno-cell.com) can be used to prop up
the coverslips and prevent cell damage, or a ring of petroleum jelly can be
painted on a microscope slide, loaded inside with serum-free media or D-PBS
and the coverslip inverted onto the gel ring, starting from one edge and slowly
lowering toward the other edge, to eliminate trapped air bubbles inside the cellmedia compartment. Non-adherent cells can be viewed by adding a drop of a
cell suspension of known concentration to a latex slide gasket mounted onto a
clean microscope slide, then covering with a coverslip.

Omega Filters+

Chroma Filters+

XF136, XF06

31000, 31013

XF100, XF23

31001, 41001

Notes
BANDPASS FILTERS FOR VIEWING HOECHST
33342 ALONE (EX 351, EM 461)
BANDPASS FILTERS FOR VIEWING
FLUORESCEIN ALONE (EX 494, EM 518)

Omega filters are supplied by Omega Optical, Inc.; Chroma filters are supplied by Chroma
Technology Corp.

V. NOTES
Buffer
The suggested tissue culture buffer, D-PBS (200mg/L KCl, 200mg/L KH2PO4,
8000mg/L NaCl, and 1150mg/L Na2HPO4) or any standard saline buffer can be
used, but color additives should be checked for fluorescence interference.
Dulbeccos Modified Eagles Medium (DMEM) or RPMI-1640 containing fetal
bovine serum (FBS) or bovine calf serum (CS) at 10% concentration, have been
used successfully in this assay format.
ROS generation
ROS have been generated in live cells via a number of methods, including 1Methyl-4-phenylpyridinium, nickel, iron, and light (UV-B and 633 nm). Please
see the references and related literature for details. You must be careful to not
photobleach or activate the DCF-DA itself in dye-loaded cells when inducing
ROS via light. We suggest include a no-cell control. Also, utilizing light outside
the absorbance spectrum of DCF-DA will minimize this potential issue;
wavelengths below 450 and above 530 nm should be ok, depending on the
intensity and duration of light exposure.
Unless otherwise indicated, these products are intended for research use only
and are not to be used for any other purposes including, but not limited to,
unauthorized commercial purposes, in vitro diagnostic purposes, ex vivo or in
vivo therapeutic purposes, investigational use in foods, drugs, devices or
cosmetics of any kind, or for consumption by or use in connection with or
administration or application to humans or animals. The products contained in
this kit have not been tested by or for us for safety or efficacy, unless expressly
stated in our catalog, on the labeling thereof, or in other documentation
accompanying the goods. These products must not be used for any other use
than research and or development. The end-user agrees to conduct all
necessary tests, comply with all applicable state and federal regulatory
requirements, issue all appropriate warnings and information to users or
subsequent users of these reagents and be responsible for storage and use by
and in accordance with the practices of a reasonable person who is an expert in
the field.

REFERENCES
1.)

2.)

3.)

4.)

5.)
6.)

7.)
8.)

9.)

10.)

11.)

12.)
13.)

14.)

Lyons TA, Amouretti XF, Held PG, Naleway JJ (2010) "Development of a LiveCell Based Reactive Oxygen Species (ROS) Assay for use in High-Content
Screening of Drug Candidates Using the BioTek Synergy Mx Microplate Reader."
presented at the Society for Biomolecular Sciences 16th Annual Conference in
Phoenix, AZ April 11-15, 2010. www.markergene.com/publications/ROS poster
for SBS 2010.pdf
Kitabchi AE, Stentz FB, Umpierrez GE. (2004) "Diabetic ketoacidosis induces in
vivo activation of human T-lymphocytes." Biochem Biophys Res Commun 315:
404-7.
Delogu G, Antonucci A, Moretti S, Marandola M, Tellan G, Signore M, Famularo
G. (2004) "Oxidative stress and mitochondrial glutathione in human lymphocytes
exposed to clinically relevant anesthetic drug concentrations." J Clin Anesth 16:
189-94.
Kalivendi S.V., Kotamraju S., Cunningham S., Shang T., Hillard C.J.,
Kalyanaraman B. (2003) 1-Methyl-4-phenylpyridinium (MPP+)-induced
apoptosis and mitochondrial oxidant generation: role of transferrin-receptordependent iron and hydrogen peroxide. Biochem J 371:151-64.
Chen CY, Wang YF, Lin YH, Yen SF. (2003) "Nickel-induced oxidative stress and
effect of antioxidants in human lymphocytes." Arch Toxicol 77: 123-30.
Tampo Y, Kotamraju S, Chitambar CR, Kalivendi SV, Keszler A, Joseph J,
Kalyanaraman B. (2003) "Oxidative stress-induced iron signaling is responsible
for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial
cells: role of transferrin receptor-dependent iron uptake in apoptosis." Circ. Res.
92:56-63.
He YY, Hader D. (2002)"Reactive oxygen species and UV-B: effect on
cyanobacteria." Photochem Photobiol Sci 1:729-36.
Burkitt MJ, Wardman P. (2001)"Cytochrome C is a potent catalyst of
dichlorofluorescin oxidation: implications for the role of reactive oxygen species
in apoptosis. Biochem. Biophys. Res. Commun. 282:329-333.
Blattner JR, He L, Lemasters JJ. (2001)"Screening assays for the mitochondrial
permeability transition using a fluorescence multiwell plate reader." Anal.
Biochem. 295: 220-226.
Hail N Jr, Lotan R. (2000)"Mitochondrial permeability transition is a central
coordinating event in N-(4-hydroxyphenyl)retinamide-induced apoptosis." Cancer
Epidemiol. Biomarkers Prev. 9:1293-1301.
Salata RA, Sullivan JA, Mandell GL. (1983) "Visualization of hydrogen peroxide
in living polymorphonuclear neutrophils utilizing leucodiacetyl 2',7'dichlorofluorescin: photomicrographic and microphotometric studies." Trans.
Assoc. Am. Physicians 96:375-383.
Ploem JS, ed. van Furth R. (1975) Mononuclear Phagocytes in Immunity,
Infection and Pathology (Blackwell Scientific, Oxford), pp. 405-421.
Nawrocka Bogusz H, Jaroszyk F (2011) May the variable magnetic field and
pulse red light induce synergy effects in respiratory burst of neutrophils in vitro?
Journal of Physics:Conf Series 329
Keston AS, Brandt R.(1965) "The fluorometric analysis of ultramicro quantities of
hydrogen peroxide." Anal. Biochem. 11:1-5.

M1049 KIT CONTENTS

DESCRIPTION

QUANTITY

PART NO.

STORAGE

Fluorescent Substrate Reagent

5 x 500g

1049-001

C, D

ROS Inducer

1 x 500L

1049-002

C, G

1 x 1mL

1049-003

REAGENTS

Buffer Solution (DMSO)

DOCUMENTATION

MSDS Sheets
Product Information Sheet

3
1

Notes: F=store at or below -20 C; C=store cold (4 C); D=store desiccated ; L=light
sensitive; T=avoid repeat freeze/thaw; R=read protocol;instructions carefully prior
to use; G=wear protective clothing/gloves/safety glasses when using.

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CONTACT AND SUPPORT

For questions or comments on this or any product from Marker
Gene Technologies, Inc., you may contact us by phone or via our website.
We welcome customer feedback and we make every effort to improve our
products based on input from our clients.
To ask a question or make a comment or suggestion, you can call
us at 1-888-218-4062 or fax to 541-342-1960.
For more information on our products and services, please visit our
website at www.markergene.com, where you can find:
Secure online ordering
Product Information
MGT Scientific Newsletters
Corporate Information
Custom Synthesis Info
We want to thank you for your purchase and hope that you will
continue to order from Marker Gene Technologies, Inc.

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Marker Gene Technologies, Inc.

University of Oregon Riverfront Research Park
1850 Millrace Drive
Eugene, Oregon 97403
1-888-218-4062
www.markergene.com

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